Long-acting polypeptides and methods of producing same

ABSTRACT

A polypeptide and polynucleotides encoding same comprising one carboxy-terminal peptide (CTP) of chorionic gonadotrophin attached to an amino terminus of a cytokine and two carboxy-terminal peptides (CTP) of chorionic gonadotrophin attached to a carboxy terminus of a cytokine are disclosed. Pharmaceutical compositions comprising the polypeptide and polynucleotides of the invention and methods of using same are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a divisional of U.S. patent application Ser. No.12/476,916, filed Jun. 2, 2009, which is a continuation-in-part of U.S.Pat. application Ser. No. 12/401,746, filed Mar. 11, 2009, which is acontinuation of U.S. patent application Ser. No. 11/700,910, filed Feb.1, 2007, which claims priority from U.S. Provisional Application Ser.No. 60/764,761, filed Feb. 3, 2006, all of which are hereby incorporatedin their entirety by reference herein.

FIELD OF INVENTION

A polypeptide and polynucleotides encoding same comprising at leastthree carboxy-terminal peptides (CTP) of chorionic gonadotrophinattached to a cytokine are disclosed. Pharmaceutical compositionscomprising the polypeptide and polynucleotides of the invention andmethods of using same are also disclosed.

BACKGROUND OF THE INVENTION

Polypeptides are susceptible to denaturation or enzymatic degradation inthe blood, liver or kidney. Accordingly, polypeptides typically haveshort circulatory half-lives of several hours. Because of their lowstability, peptide drugs are usually delivered in a sustained frequencyso as to maintain an effective plasma concentration of the activepeptide. Moreover, since peptide drugs are usually administered byinfusion, frequent injection of peptide drugs causes considerablediscomfort to a subject. Thus, there is a need for technologies thatwill prolong the half-lives of therapeutic polypeptides whilemaintaining a high pharmacological efficacy thereof. Such desiredpeptide drugs should also meet the requirements of enhanced serumstability, high activity and a low probability of inducing an undesiredimmune response when injected into a subject.

Unfavorable pharmacokinetics, such as a short serum half-life, canprevent the pharmaceutical development of many otherwise promising drugcandidates. Serum half-life is an empirical characteristic of amolecule, and must be determined experimentally for each new potentialdrug. For example, with lower molecular weight polypeptide drugs,physiological clearance mechanisms such as renal filtration can make themaintenance of therapeutic levels of a drug unfeasible because of costor frequency of the required dosing regimen. Conversely, a long serumhalf-life is undesirable where a drug or its metabolites have toxic sideeffects.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a polypeptidecomprising a cytokine, one chorionic gonadotrophin carboxy terminalpeptide (CTP) attached to an amino terminus of the cytokine, and twochorionic gonadotrophin carboxy terminal peptides attached to a carboxyterminus of the cytokine.

In another embodiment, the present invention further provides apolynucleotide comprising a coding portion encoding a polypeptide,wherein the polypeptide comprises a cytokine, one chorionicgonadotrophin carboxy terminal peptide (CTP) attached to an aminoterminus of the cytokine, and two chorionic gonadotrophin carboxyterminal peptides attached to a carboxy terminus of the cytokine.

In another embodiment, the present invention further provides a methodof reducing a dosing frequency of a cytokine, comprising the step ofattaching one chorionic gonadotrophin carboxy terminal peptide to anamino terminus of the cytokine and two chorionic gonadotrophin carboxyterminal peptides to a carboxy terminus of the cytokine, therebyreducing a dosing frequency of a cytokine.

In another embodiment, the present invention further provides a methodof increasing compliance in the use of cytokine therapy, comprisingproviding to a subject in need thereof, a polypeptide comprising acytokine, one chorionic gonadotrophin carboxy terminal peptide (CTP)attached to an amino terminus of the cytokine, and two chorionicgonadotrophin carboxy terminal peptides attached to a carboxy terminusof the cytokine, thereby increasing compliance in the use of cytokinetherapy.

In another embodiment, the present invention further provides a methodof improving a biological half life of a cytokine, comprising the stepof attaching one chorionic gonadotrophin carboxy terminal peptide to anamino terminus of the cytokine and two chorionic gonadotrophin carboxyterminal peptides to a carboxy terminus of the cytokine, therebyimproving a biological half life of a cytokine.

In another embodiment, the present invention further provides a methodof improving the area under the curve (AUC) of a cytokine, comprisingthe step of attaching one chorionic gonadotrophin carboxy terminalpeptide to an amino terminus of the cytokine and two chorionicgonadotrophin carboxy terminal peptides to a carboxy terminus of thecytokine, thereby improving the area under the curve (AUC) of acytokine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F are diagrams illustrating six EPO-CTP constructs.

FIG. 1A is a diagram of the polypeptide of SEQ ID NO: 1.

FIG. 1B is a diagram of the polypeptide of SEQ ID NO: 2.

FIG. 1C is a diagram of the polypeptide of SEQ ID NO: 3.

FIG. 1D is a diagram of the polypeptide of SEQ ID NO: 4.

FIG. 1E is a diagram of the polypeptide of SEQ ID NO: 5.

FIG. 1F is a diagram of the polypeptide of SEQ ID NO: 6.

FIG. 2 is a photograph illustrating the expression of the EPO-CTPvariants from transfected DG44 cells. Final test samples fromtransfected cells were prepared as described under “sample preparation”and run on SDS/PAGE. Proteins were detected by Western blot.

FIG. 3 is a graph illustrating the in vivo bioactivity of recombinanthEPO derivatives and EPO-3 (SEQ ID NO: 3). ICR mice (n=7/group) receiveda single i.v. injection/week (15 μg/kg) for three weeks of EPO-3,rhEPO-WT (SEQ ID NO: 16), Recormon® (Commercial EPO) or Recormon® (5μg/kg) 3 times a week. Control animals were injected i.v. with PBS.Blood samples were collected three times a week and haematocrit levelswere detected. Each point represents the group average of haematocrit(%) ±SE.

FIG. 4 is a graph illustrating the in vivo bioactivity of recombinanthEPO derivatives and EPO-1 (SEQ ID NO: 1). ICR mice (n=7/group) receiveda single i.v. injection/week (15 μg/kg) for three weeks of EPO-1,rhEPO-WT (SEQ ID NO: 16), Recormon® or Recormon® (5 μg/kg) 3 times aweek. Control animals were injected i.v. with PBS. Blood samples werecollected three times a week and haematocrit levels were detected. Eachpoint represents the group average of haematocrit (%) ±SE.

FIG. 5 is a graph illustrating the in vivo bioactivity of recombinanthEPO derivatives and EPO-2 (SEQ ID NO: 2). ICR mice (n=7/group) receiveda single i.v. injection/week (15 μg/kg) for three weeks of EPO-2 (SEQ IDNO: 2), rhEPO-WT (SEQ ID NO: 16), Recormon® or Recormon® (5 μg/kg) 3times a week. Control animals were injected i.v. with PBS. Blood sampleswere collected three times a week and haematocrit levels were detected.Each point represents the group average of haematocrit (%) ±SE.

FIG. 6 is a time graph illustrating the change in reticulocyte levelfollowing a single bolus dose of EPO-0 (SEQ ID NO: 16), EPO-3 (SEQ IDNO: 3) and Aranesp®.

FIG. 7 is a time graph illustrating the change in hemoglobin level(presented as change from baseline) following a single bolus dose ofEPO-0 (SEQ ID NO: 16), EPO-3 (SEQ ID NO: 3) and Aranesp®.

FIG. 8 is a time graph illustrating the change in hematocrit levelfollowing a single bolus dose of EPO-0 (SEQ ID NO: 16), EPO-3 (SEQ IDNO: 3) and Aranesp®.

FIG. 9 is a graph illustrating the change in serum concentration ofEPO-0 (SEQ ID NO: 16), EPO-3 (SEQ ID NO: 3) and Aranesp® post i.v.injection.

FIG. 10 is a Western blot illustrating the molecular weight & identityof MOD-4020 (SEQ ID NO: 36), MOD-4021 (SEQ ID NO: 37), MOD-4022 (SEQ IDNO: 38), MOD-4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40). PAGE SDSgel was blotted and stained using monoclonal anti-hGH antibodies. Thephotograph indicates that like commercial and wild type hGH, MOD-7020-4variants are recognized by anti-hGH antibodies.

FIG. 11 is a bar graph illustrating the weight gain of hypophysectomizedrats following administration of the GH-CTP polypeptides of the presentinvention.

FIG. 12 is a Western blot illustrating the molecular weight & identityof Avonex®, MOD-9013 (SEQ ID NO: 56), MOD-9016 (SEQ ID NO: 62), MOD-9015(SEQ ID NO: 60), MOD-9012 (SEQ ID NO: 54), MOD-9011 (SEQ ID NO: 52) andMock. PAGE SDS gel was blotted and stained using monoclonal anti-IFN-β1Aantibodies (B). The photograph indicates that like commercial Avonex®,MOD-901X variants are recognized by anti IFN-β1A antibodies.

FIG. 13. Mean plasma IFNβ1a or MOD-901X varients concentrations (ng/ml)following single-dose i.v. administration of IFN-β1a or MOD-901Xvarients in SD rats (n=3 per dose/route/timepoint). IFN-β1a serumconcentrations were determined using commercial ELISA kit.

FIG. 14. MOD-9010 amino acid sequence (SEQ ID NO: 48) and nucleic acidsequence (SEQ ID NO: 49), (A), MOD-9011 amino acid sequence (SEQ ID NO:52) and nucleic acid sequence (SEQ ID NO: 53), (B), MOD-9012 amino acidsequence (SEQ ID NO: 54) and nucleic acid sequence (SEQ ID NO: 55), (C),MOD-9013 amino acid sequence (SEQ ID NO: 56) and nucleic acid sequence(SEQ ID NO: 57), (D), MOD-9014 amino acid sequence (SEQ ID NO: 58) andnucleic acid sequence (SEQ ID NO: 59), (E), MOD-9015 amino acid sequence(SEQ ID NO: 60) and nucleic acid sequence (SEQ ID NO: 61), (F), andMOD-9016 amino acid sequence (SEQ ID NO: 62) and nucleic acid sequence(SEQ ID NO: 63), (G) amino acid (AA) sequences followed by DNAsequences. Underline: Signal sequence, Black letters: Mature protein,Italic: CTP unit.

FIG. 15. are graphs showing the mean plasma concentrations (ng/ml) ofRebif®, MOD-9012, and MOD-9013 following single-dose i.v. or s.c.administration of IFN-β1a or MOD-9012, and MOD-9013 in SD rats (n=3 perdose/route/timepoint). IFN-β1a serum concentrations were determinedusing a commercial ELISA kit.

FIG. 16 includes two schemes (1) a map of MOD 4023 pCI-dhfr Plasmid and(2) structural protein formula of MOD-4023.

FIG. 17 are graphs showing the mean plasma MOD-4023 or GH concentrations(pg/ml) following a single i.v. or s.c. dose of MOD-4023 or GH in rats(n=3-6 per dose/route).

FIG. 18 is a graph showing the mean incremental weight gain following asingle s.c. doses of MOD-4023 (0.4, 0.8 and 4 mg/Kg) inhypophysectomized rats in comparison to daily GH injections (0.1mg/Kg/Day) (n=10 per dose).

FIG. 19 is a graph showing the area under the curve following singleinjection of MOD-4023 correlates with body weight gain in rats.

FIG. 20 is a graph showing the incremental weight gain following a s.c.doses of MOD-4023 (0.4, 0.8 and 4 mg/Kg) 4 days apart inhypophysectomized rats in comparison to daily GH injections (0.1mg/Kg/Day) (n=10 per dose).

FIG. 21 is a graph showing hGH serum concentration in hypophysectomizedrats following s.c. injection of MOD-4023 and commercial hGH. Singledose of MOD-4023 0.6 or 1.8 mg/Kg and Biotropin® 0.35 or 1.05 mg/Kg wereinjected subcutaneously to hypophysectomised rats for determination ofPK/PD profile. Serum hGH post injection was measured using specificELISA kits.

FIG. 22 is a graph showing IGF-1 serum levels in hypophysectomized ratsFollowing s.c. injection of MOD-4023 and commercial hGH. Single dose ofMOD-4023 0.6 or 1.8 mg/Kg and Biotropin® 0.35 or 1.05 mg/Kg wereinjected subcutaneously to hypophysectomised rats for determination ofPK/PD profile. Serum IGF-I post injection was measured using specificELISA kits (Roche Diagnostics).

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides long-acting cytokinesand methods of producing and using same. In another embodiment,long-acting cytokines comprise carboxy terminal peptide (CTP, alsoreferred to as CTP unit). In another embodiment, long-actingpolypeptides comprise carboxy terminal peptide (CTP) of human ChorionicGonadotropin (hCG). In another embodiment, CTP acts as a protectantagainst degradation of cytokines or polypeptides of interest. In anotherembodiment, CTP extends the C_(max) of cytokines or polypeptides ofinterest. In another embodiment, CTP extends the T_(max) of cytokines orpolypeptides of interest. In another embodiment, CTP extends circulatoryhalf-lives of cytokines or polypeptides of interest. In someembodiments, CTP enhances the potency of cytokines or polypeptides ofinterest.

In other embodiments, engineered cytokines or polypeptides of interestof the invention comprising a single CTP attached to the amino terminusand two CTP peptides attached in tandem to the carboxy terminus are atleast equivalent to the non CTP-modified cytokines or polypeptides ofinterest, in terms of biological activity. In other embodiments,engineered cytokines or polypeptides of interest of the inventioncomprising a single CTP attached to the amino terminus and two CTPpeptides attached in tandem to the carboxy terminus are at leastequivalent to the non CTP-modified cytokines or polypeptides ofinterest, in terms of pharmacological measures such as pharmacokineticsand pharmacodynamics.

In another embodiment, the present invention provides a polypeptidecomprising a cytokine and at least one CTP peptide attached to an aminoterminus of the cytokine and at least two chorionic gonadotrophincarboxy terminal peptides attached to a carboxy terminus of thecytokine. In another embodiment, the present invention provides apolypeptide comprising one chorionic gonadotrophin carboxy terminalpeptide attached to an amino terminus of a cytokine and two chorionicgonadotrophin carboxy terminal peptides attached to a carboxy terminusof a cytokine.

In another embodiment, the terms “CTP peptide,” “carboxy terminalpeptide” and “CTP sequence” are used interchangeably herein. In anotherembodiment, the carboxy terminal peptide is a full-length CTP. Inanother embodiment, the carboxy terminal peptide is a truncated CTP.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, “signal sequence” and “signal peptide” are usedinterchangeably herein. In another embodiment, “sequence” when inreference to a polynucleotide can refer to a coding portion. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the invention provides a polypeptide consistingof a cytokine, a single chorionic gonadotrophin carboxy terminal peptideattached to the amino terminus of the cytokine, and two chorionicgonadotrophin carboxy terminal peptides attached to the carboxy terminusof the cytokine. In another embodiment, the invention provides apolypeptide consisting of a cytokine, a single chorionic gonadotrophincarboxy terminal peptide attached to the amino terminus of the cytokine,two chorionic gonadotrophin carboxy terminal peptides attached to thecarboxy terminus of the cytokine, and a signal peptide attached to theamino terminus of one chorionic gonadotrophin carboxy terminal peptide.

In another embodiment, a cytokine is a low molecular weight protein. Inanother embodiment, a cytokine is a protein secreted by a cell. Inanother embodiment, a cytokine induces and/or regulates an immuneresponse. In another embodiment, a cytokine has a high affinity bindingto a specific receptor or receptors. In another embodiment, cytokines asdescribed herein include mimetics of cytokines that can be used toinhibit or potentiate their effects in vivo. In another embodiment, acytokine comprises an autocrine activity. In another embodiment, acytokine comprises a paracrine activity. In another embodiment, acytokine comprises an endocrine activity.

In another embodiment, the cytokine is a Hematopoietin cytokine. Inanother embodiment, the cytokine is an Interferon cytokine. In anotherembodiment, the cytokine is a chemokine. In another embodiment, thecytokine is a Tumor Necrosis Factor cytokine. In another embodiment, acytokine as used herein comprises biological activity and clinicalefficacy. In another embodiment, a cytokine as used herein is atherapeutic protein.

In another embodiment, a cytokine comprising CTPs as described hereinhas enhanced biological activity in vivo compared to the same cytokinewithout CTPs. In another embodiment, a cytokine comprising at least oneCTP attached to its amino terminus and at least two CTPs attached to itscarboxy terminus has enhanced biological activity in vivo compared tothe same cytokine without CTPs. In another embodiment, a cytokinecomprising one CTP attached to its amino terminus and two CTPs attachedto its carboxy terminus has enhanced biological in vivo activitycompared to the same cytokine in without CTPs.

In another embodiment, a cytokine modified with CTPs is used tofacilitate organ transplantation. In another embodiment, a cytokinemodified with CTPs is used to reduce inflammation. In anotherembodiment, a cytokine modified with CTPs is used to induceerythropoiesis. In another embodiment, a cytokine modified with CTPs isused to induce growth. In another embodiment, a cytokine modified withCTPs is used to induce weight gain. In another embodiment, a cytokinemodified with CTPs is used in cancer therapy as will be readilyunderstood by one of average skill in the art. In another embodiment, acytokine modified with CTPs is used to induce an immune response. Inanother embodiment, a cytokine modified with CTPs is used in infectiousdisease therapy as will be readily understood by one of average skill inthe art. In another embodiment, a cytokine modified with CTPs is used intreating allergy as will be readily understood by one of average skillin the art.

In another embodiment, a subject is a human subject. In anotherembodiment, a subject is a pet. In another embodiment, a subject is amammal. In another embodiment, a subject is a farm animal. In anotherembodiment, a subject is a monkey. In another embodiment, a subject is ahorse. In another embodiment, a subject is a cow. In another embodiment,a subject is a mouse. In another embodiment, a subject is a rat.

In another embodiment, a CTP-cytokine-CTP-CTP as described hereincomprises a cytokine or an active fragment thereof connected via apeptide bond to at least one CTP unit. In another embodiment, aCTP-cytokine-CTP-CTP as described herein comprises a cytokine or anactive fragment thereof connected via a peptide bond to at least one CTPunit which is connected to an additional CTP unit via a peptide bond. Inanother embodiment, a polypeptide as described herein comprisingcytokine fragments thereof, and CTP units and/or fragments thereof areinterconnected via a peptide bond. In another embodiment, one nucleicacid molecule encodes a polypeptide as described herein comprising acytokine and/or fragments thereof, and CTP units and/or fragmentsthereof.

In one embodiment, the cytokine is a homologue. In one embodiment, ahomologue also refers to a deletion, insertion, or substitution variant,including an amino acid substitution thereof, and biologically activepolypeptide fragments thereof.

In another embodiment, the invention provides a polypeptide consistingof a cytokine antagonist, a single chorionic gonadotrophin carboxyterminal peptide attached to the amino terminus of the cytokineantagonist, and two chorionic gonadotrophin carboxy terminal peptidesattached to the carboxy terminus of the cytokine antagonist. In anotherembodiment, the invention provides a polypeptide consisting of acytokine antagonist, a single chorionic gonadotrophin carboxy terminalpeptide attached to the amino terminus of the cytokine antagonist, twochorionic gonadotrophin carboxy terminal peptides attached to thecarboxy terminus of the cytokine antagonist, and a signal peptideattached to the amino terminus of one chorionic gonadotrophin carboxyterminal peptide.

In another embodiment, a cytokine antagonist modified with CTPs isapplied as an anti-cytokine strategy. In another embodiment, a cytokineantagonist modified with CTPs is effective in decreasing an inflammatoryresponse. In another embodiment, a cytokine antagonist modified withCTPs is more effective in decreasing an inflammatory response comparedto an unmodified cytokine antagonist. In another embodiment, a cytokineantagonist modified with CTPs is more stable than an unmodified cytokineantagonist. In another embodiment, a cytokine antagonist modified withCTPs is more stable in vivo than an unmodified cytokine antagonist. Inanother embodiment, a cytokine antagonist modified with CTPs is morebioactive than an unmodified cytokine antagonist.

In another embodiment, a cytokine antagonist is a cytokine homologue. Inanother embodiment, a cytokine antagonist is a soluble fragment of acytokine receptor. In another embodiment, a cytokine antagonist is achemokine receptor homologue.

In another embodiment, a cytokine as described herein is involved incytokine signaling cascade comprising Ras-MAP kinase pathway. In anotherembodiment, a cytokine as described herein is involved in induction ofJNK. In another embodiment, a cytokine as described herein is involvedin induction of p38MAP. In another embodiment, a cytokine as describedherein induces cell proliferation. In another embodiment, a cytokine asdescribed herein is involved in cytokine signaling cascade comprisingthe JAK/STAT pathway. In another embodiment, a cytokine as describedherein induces cell growth inhibition. In another embodiment, a cytokineas described herein induces differentiation.

In another embodiment, a cytokine as described herein is a four α-helixbundle cytokine. In another embodiment, a cytokine as described hereinis a long-chain 4-helix bundle cytokine. In another embodiment, acytokine as described herein is a short-chain 4-helix bundle cytokine.

In another embodiment, a cytokine as described herein is a beta-trefoilcytokine. In another embodiment, a cytokine as described herein is abeta-sandwich cytokine. In another embodiment, a cytokine as describedherein is an EGF-like cytokine. In another embodiment, a cytokine asdescribed herein comprises a Cystine knot dimerization domain. Inanother embodiment, a cytokine as described herein comprises both alphaand beta chains. In another embodiment, a cytokine as described hereinis an alpha superfamily cytokine such as IL-2, IL-4, IL-5, GM-CSF, IL-3,IFN-alpha, or IL-13. In another embodiment, a cytokine as describedherein is a dimeric 4-helix bundles cytokine. In another embodiment, acytokine as described herein is a member of the IL family of cytokines.

In another embodiment, a cytokine as described herein is a long-chain4-helix bundle superfamily cytokine such as GH, G-CSF, Myelomonocyticgrowth factor, IL-6, IL-3, IL-7, LIF, Oncostatin M, Ciliary neurotrophicfactor (CNTF), or cholinergic differentiation factor (CDF). In anotherembodiment, a cytokine as described herein is a short-chain 4-helixbundle superfamily cytokine such as IL-2, IL-4, IL-13, IFN-alpha, IL-5,GM-CSF, IL-3, or Macrophage colony-stimulating factor (M-CSF). Inanother embodiment, a cytokine as described herein is a dimeric 4-helixbundles such as IFN-Gamma, IL-10, or IFN-Beta.

In another embodiment, a cytokine as described herein is a Beta-Trefoilcytokine such as IL1-A, IL1-B, or FGF. In another embodiment, a cytokineas described herein is a Beta-sandwich cytokine such as TNF-alpha orTNF-Beta. In another embodiment, a cytokine as described herein is anEGF-like cytokine such as TGF-Alpha. In another embodiment, a cytokineas described herein comprises cystine knot dimerization domains. Inanother embodiment, Gonadotropin, Nerve Growth Factor (NGF),Platelet-derived growth factor (PDGF), and TGF-Beta2 comprise cystineknot dimerization domains. In another embodiment, a cytokine asdescribed herein comprises both alpha and beta chains. In anotherembodiment, IL-8, IP10, platelet factor 4 (PF-4), bTG, GRO, 9E3, HLA-A2,macrophage inflammatory protein 1 alpha (MIP-1 alpha), macrophageinflammatory protein 1 beta (MW-1 beta), and Melanoma growth stimulatingactivity (MGSA) comprise both alpha and beta chains.

In another embodiment, a cytokine as described herein binds ahematopoietin-receptor family member (also called the class I cytokinereceptor family). In another embodiment, a cytokine as described hereinbinds a class II cytokine receptor (interferons or interferon-likecytokines). In another embodiment, a cytokine as described herein bindsa tumor necrosis factor-receptor (TNFR). In another embodiment, acytokine as described herein binds a chemokine receptor. In anotherembodiment, a cytokine as described herein binds a G protein-coupledreceptor.

In another embodiment, a cytokine as described herein is an IL-2cytokine. In another embodiment, a cytokine as described herein is aninterferon. In another embodiment, a cytokine as described herein is anIL-10 cytokine. In another embodiment, a cytokine as described herein isEPO. In another embodiment, a cytokine as described herein isthrombopoietin (THPO). In another embodiment, a cytokine as describedherein is IL-1, IL-18, or IL-17. In another embodiment, a cytokine asdescribed herein promotes proliferation of T-cells.

In another embodiment, a cytokine as described herein is a member of thesuperfamily of growth hormone (GH)-like cytokines. In anotherembodiment, a cytokine as described herein is close to the clusterformed by ciliary neurotrophic factor and granulocyte colony-stimulatingfactor (CSF).

In another embodiment, a cytokine as described herein enhances cytokineresponses, type 1 (IFN-γ, TGF-βetc.). In another embodiment, a cytokineas described herein enhances antibody responses, type 2 (IL-4, IL-10,IL-13, etc).

In another embodiment, a cytokine is a peptide. In another embodiment,the cytokine is glycosylated. In another embodiment, a cytokine is apolypeptide. In another embodiment, a cytokine as described herein is amodified cytokine comprising at least one CTP peptide attached to anamino terminus of said cytokine and at least two chorionic gonadotrophincarboxy terminal peptides attached to a carboxy terminus of saidcytokine. In another embodiment, a cytokine as described herein is amodified cytokine consisting of a cytokine, one CTP peptide attached toan amino terminus of the cytokine, and at least two chorionicgonadotrophin carboxy terminal peptides attached to a carboxy terminusof the cytokine. In another embodiment, a cytokine as described hereinis a modified cytokine consisting of a cytokine, at least one CTPpeptide attached to an amino terminus of the cytokine, and two chorionicgonadotrophin carboxy terminal peptides attached to a carboxy terminusof the cytokine. In another embodiment, a cytokine as described hereinis a modified cytokine consisting of a cytokine, one CTP peptideattached to an amino terminus of the cytokine, and two chorionicgonadotrophin carboxy terminal peptides attached to a carboxy terminusof the cytokine.

In another embodiment, the carboxy-terminal peptide (CTP) is attached tothe cytokine via a linker. In another embodiment, the linker whichconnects the CTP sequence to the cytokine is a covalent bond. In anotherembodiment, the linker which connects the CTP sequence to the cytokineis a peptide bond. In another embodiment, the linker which connects theCTP sequence to the cytokine is a substituted peptide bond. In anotherembodiment, the carboxy-terminal peptide (CTP) sequence comprises anamino acid sequence selected from the sequences set forth in SEQ ID NO:17 and SEQ ID NO: 18.

In another embodiment, SEQ ID NO: 17 comprise the following amino acid(AA) sequence: DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 17). Inanother embodiment, SEQ ID NO: 18 comprise the following amino acid (AA)sequence: SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 18).

In another embodiment, the carboxy terminal peptide (CTP) peptide of thepresent invention comprises the amino acid sequence from amino acid 112to position 145 of human chorionic gonadotrophin, as set forth in SEQ IDNO: 17. In another embodiment, the CTP sequence of the present inventioncomprises the amino acid sequence from amino acid 118 to position 145 ofhuman chorionic gonadotropin, as set forth in SEQ ID NO: 18. In anotherembodiment, the CTP sequence also commences from any position betweenpositions 112-118 and terminates at position 145 of human chorionicgonadotrophin. In some embodiments, the CTP sequence peptide is 28, 29,30, 31, 32, 33 or 34 amino acids long and commences at position 112,113, 114, 115, 116, 117 or 118 of the CTP amino acid sequence.

In another embodiment, the CTP peptide is a variant of chorionicgonadotrophin CTP which differs from the native CTP by 1-5 conservativeamino acid substitutions as described in U.S. Pat. No. 5,712,122 whichis incorporated herein by reference. In another embodiment, the CTPpeptide is a variant of chorionic gonadotrophin CTP which differs fromthe native CTP by 1 conservative amino acid substitution. In anotherembodiment, the CTP peptide is a variant of chorionic gonadotrophin CTPwhich differs from the native CTP by 2 conservative amino acidsubstitutions. In another embodiment, the CTP peptide is a variant ofchorionic gonadotrophin CTP which differs from the native CTP by 3conservative amino acid substitutions. In another embodiment, the CTPpeptide is a variant of chorionic gonadotrophin CTP which differs fromthe native CTP by 4 conservative amino acid substitutions. In anotherembodiment, the CTP peptide is a variant of chorionic gonadotrophin CTPwhich differs from the native CTP by 5 conservative amino acidsubstitutions. In another embodiment, the CTP peptide amino acidsequence of the present invention is at least 70% homologous to thenative CTP amino acid sequence or a peptide thereof. In anotherembodiment, the CTP peptide amino acid sequence of the present inventionis at least 80% homologous to the native CTP amino acid sequence or apeptide thereof. In another embodiment, the CTP peptide amino acidsequence of the present invention is at least 90% homologous to thenative CTP amino acid sequence or a peptide thereof. In anotherembodiment, the CTP peptide amino acid sequence of the present inventionis at least 95% homologous to the native CTP amino acid sequence or apeptide thereof.

In another embodiment, the CTP peptide DNA sequence of the presentinvention is at least 70% homologous to the native human CTP DNAsequence or a peptide thereof. In another embodiment, the CTP peptideDNA sequence of the present invention is at least 80% homologous to thenative human CTP DNA sequence or a peptide thereof. In anotherembodiment, the CTP peptide DNA sequence of the present invention is atleast 90% homologous to the native CTP DNA sequence or a peptidethereof. In another embodiment, the CTP peptide DNA sequence of thepresent invention is at least 95% homologous to the native CTP DNAsequence or a peptide thereof.

In one embodiment, at least one of the chorionic gonadotrophin CTP aminoacid sequences is truncated. In another embodiment, both of thechorionic gonadotrophin CTP amino acid sequences are truncated. Inanother embodiment, 2 of the chorionic gonadotrophin CTP amino acidsequences are truncated. In another embodiment, 2 or more of thechorionic gonadotrophin CTP amino acid sequences are truncated. Inanother embodiment, all of the chorionic gonadotrophin CTP amino acidsequences are truncated. In one embodiment, the truncated CTP comprisesthe first 10 amino acids of SEQ ID NO: 67. In another embodiment, SEQ IDNO: 67 comprises the following amino acid (AA) sequence: SSSSKAPPPSLP.

In one embodiment, the truncated CTP comprises the first 11 amino acidsof SEQ ID NO:43. In one embodiment, the truncated CTP comprises thefirst 12 amino acids of SEQ ID NO: 67. In one embodiment, the truncatedCTP comprises the first 8 amino acids of SEQ ID NO: 67. In oneembodiment, the truncated CTP comprises the first 13 amino acids of SEQID NO: 43. In one embodiment, the truncated CTP comprises the first 14amino acids of SEQ ID NO: 67. In one embodiment, the truncated CTPcomprises the first 6 amino acids of SEQ ID NO: 67. In one embodiment,the truncated CTP comprises the first 5 amino acids of SEQ ID NO: 67.

In one embodiment, at least one of the chorionic gonadotrophin CTP aminoacid sequences is glycosylated. In another embodiment, both of thechorionic gonadotrophin CTP amino acid sequences are glycosylated. Inanother embodiment, 2 of the chorionic gonadotrophin CTP amino acidsequences are glycosylated. In another embodiment, 2 or more of thechorionic gonadotrophin CTP amino acid sequences are glycosylated. Inanother embodiment, all of the chorionic gonadotrophin CTP amino acidsequences are glycosylated. In one embodiment, the CTP sequence of thepresent invention comprises at least one glycosylation site. In oneembodiment, the CTP sequence of the present invention comprises 2glycosylation sites. In one embodiment, the CTP sequence of the presentinvention comprises 3 glycosylation sites. In one embodiment, the CTPsequence of the present invention comprises 4 glycosylation sites.

In another embodiment, at least one carboxy-terminal peptide (CTP)sequence comprises an amino acid sequence selected from the sequencesset forth in SEQ ID NO: 17 and SEQ ID NO: 18. In another embodiment, atleast one carboxy-terminal peptide (CTP) is truncated.

In another embodiment, the cytokine further comprises a signal peptidefor its secretion. In some embodiments, signal sequences include, butare not limited to the endogenous signal sequence for IFN. In someembodiments, signal sequences include, but are not limited to theendogenous signal sequence of any known cytokine. In another embodiment,the polypeptides and methods of the present invention provide a cytokinehaving additionally a signal peptide of SEQ ID NO: 64 and at least oneCTP peptide on the N-terminus and at least one CTP peptide on theC-terminus. In another embodiment, the polypeptides and methods of thepresent invention provide a cytokine having additionally on theN-terminus the signal peptide of SEQ ID NO: 64 and at least one CTPpeptide on the N-terminus and at least two CTP peptides on theC-terminus. In another embodiment, the polypeptides and methods of thepresent invention provide a cytokine having additionally on theN-terminus the signal peptide of SEQ ID NO: 64 and a single CTP peptideon the N-terminus and two CTP peptides on the C-terminus. In anotherembodiment, SEQ ID NO: 64 comprise the following amino acid (AA)sequence: MTNKCLLQIALLLCFSTTALS (SEQ ID NO: 64).

In some embodiments, CTP sequences at both the amino terminal end of acytokine and at the carboxy terminal end of the cytokine provideenhanced protection against degradation of a cytokine. In anotherembodiment, at least one CTP sequence at the amino terminal end of acytokine and two CTP units at the carboxy terminal end of a cytokineprovide enhanced protection against clearance. In another embodiment, atleast one CTP sequence at the amino terminal end of a cytokine and twoCTP units at the carboxy terminal end of a cytokine provide prolongedclearance time. In another embodiment, at least one CTP sequence at theamino terminal end of a cytokine and two CTP units at the carboxyterminal end of a cytokine enhance C_(max) of a cytokine. In anotherembodiment, at least one CTP sequence at the amino terminal end of acytokine and two CTP units at the carboxy terminal end of a cytokineenhance T_(max) of a cytokine. In another embodiment, at least one CTPsequence at the amino terminal end of a cytokine and two CTP units atthe carboxy terminal end of a cytokine enhanced T_(1/2).

In some embodiments, CTP sequences at both the amino terminal end of acytokine and at the carboxy terminal end of the cytokine extend thehalf-life of the attached cytokine. In another embodiment, at least asingle CTP sequence at the amino terminal end of a cytokine and at leasttwo CTP sequences at the carboxy terminal end of the cytokine provide anextended half-life to the modified cytokine. In another embodiment, asingle CTP sequence at the amino terminal end of a cytokine and two CTPsequences at the carboxy terminal end of the cytokine provide anextended half-life to the attached cytokine. In another embodiment, asingle CTP sequence at the amino terminal end of a cytokine and two CTPsequences in tandem at the carboxy terminal end of the cytokine providean extended half-life to the cytokine.

In some embodiments, a CTP sequence at the amino terminal end of apolypeptide, a CTP sequence at the carboxy terminal end of the cytokine,and at least one additional CTP sequence attached in tandem to the CTPsequence at the carboxy terminus provide enhanced protection againstdegradation to a cytokine. In some embodiments, a CTP sequence at theamino terminal end of a cytokine, a CTP sequence at the carboxy terminalend of the cytokine, and at least one additional CTP sequence attachedin tandem to the CTP sequence at the carboxy terminus extend thehalf-life of the cytokine. In some embodiments, a CTP sequence at theamino terminal end of a cytokine, a CTP sequence at the carboxy terminalend of the cytokine, and at least one additional CTP sequence attachedin tandem to the CTP sequence at the carboxy terminus enhance thebiological activity of the cytokine.

In another embodiment, conjugated cytokines of this invention are usedin the same manner as unmodified cytokines. In another embodiment,conjugated cytokines of this invention have an increased circulatinghalf-life and plasma residence time, decreased clearance, and increasedclinical activity in vivo. In another embodiment, due to the improvedproperties of the conjugated cytokines as described herein, theseconjugates are administered less frequently than unmodified cytokines.In another embodiment, conjugated cytokines as described herein areadministered once a week instead of the three times a week forunmodified cytokines. In another embodiment, decreased frequency ofadministration will result in improved patient compliance leading toimproved treatment outcomes, as well as improved patient quality oflife. In another embodiment, compared to conventional conjugates ofcytokines linked to poly(ethylene glycol), it has been found thatconjugates having the molecular weight and linker structure of theconjugates of this invention have an improved potency, improvedstability, elevated AUC levels, and enhanced circulating half-life. Inanother embodiment, compared to conventional conjugates of cytokineslinked to poly(ethylene glycol), it has been found that EPO having themolecular weight and linker structure of the conjugates of thisinvention have an improved potency, improved stability, elevated AUClevels, and enhanced circulating half-life. In another embodiment, atherapeutically effective amount of a conjugated cytokine is the amountof conjugate necessary for the in vivo measurable expected biologicalactivity. In another embodiment, a therapeutically effective amount of aconjugated EPO is the amount of EPO conjugate necessary for thebiological activity of inducing bone marrow cells to increase productionof reticulocytes and red blood cells. In another embodiment, atherapeutically effective amount of a conjugated cytokine is determinedaccording to factors as the exact type of condition being treated, thecondition of the patient being treated, as well as the other ingredientsin the composition. In another embodiment, a therapeutically effectiveamount of a conjugated cytokine is 0.01 to 10 μg per kg body weightadministered once a week. In another embodiment, a therapeuticallyeffective amount of a conjugated cytokine is 0.1 to 1 μg per kg bodyweight, administered once a week. In another embodiment, apharmaceutical composition comprising a conjugated cytokine isformulated at a strength effective for administration by various meansto a human patient.

In another embodiment, the cytokine is an interferon. In anotherembodiment, the IFN amino acid sequence of the present invention is atleast 60% homologous to an IFN sequence set forth in GenBank AccessionNo. NP_(—)002167.1 as determined using BlastP software of the NationalCenter of Biotechnology Information (NCBI) using default parameters. Inanother embodiment, the IFN amino acid sequence of the present inventionis at least 70% homologous to an IFN sequence set forth in GenBankAccession No. NP_(—)002167.1 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters. In another embodiment, the IFN amino acid sequence of thepresent invention is at least 80% homologous to an IFN sequence setforth in GenBank Accession No. NP_(—)002167.1 as determined using BlastPsoftware of the National Center of Biotechnology Information (NCBI)using default parameters. In another embodiment, the IFN amino acidsequence of the present invention is at least 90% homologous to an IFNsequence set forth in GenBank Accession No. NP_(—)002167.1 as determinedusing BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters.

In some embodiments, “homology” according to the present invention alsoencompasses deletions, insertions, or substitution variants, includingan amino acid substitution thereof, and biologically active polypeptidefragments thereof. In one embodiment, the substitution variant is one inwhich the glutamine in position 65 of hGH is substituted by a valine(SEQ ID NO: 23) [Gellerfors et al., J Pharm Biomed Anal 1989, 7:173-83].

In another embodiment, a cytokine utilized according to the teachings ofthe present invention exhibits increased potency. In some embodiments,the attachment of CTP sequence to both the amino and carboxy termini ofa cytokine results in prolonged in vivo activity.

In one embodiment, the term “interferon” refers to the mammalianinterferon polypeptide (e.g., Type I). In one embodiment, “interferon”refers to the mammalian interferon polypeptide (Type II interferon)which exhibits an interferon activity, e.g. antiviral orantiproliferative activity. In another embodiment, GenBank AccessionNumbers of non-limiting examples of interferons are listed in Table 1below. An interferon of the present invention also refers in oneembodiment, to homologs (e.g., polypeptides which are at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 87%, at least 89%, at least 91%, atleast 93%, at least 95% or more say 100% homologous to interferonsequences listed in Table 1 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters). In another embodiment, homolog may also refer to adeletion, insertion, or substitution variant, including an amino acidsubstitution thereof, and biologically active polypeptide fragmentsthereof. In some embodiments, additional suitable interferonpolypeptides are as known to those of ordinary skill in the art. In someembodiments, the interferon is a Type I or Type II interferon, includingthose commonly designated as alpha-interferon. In some embodiments, theinterferon is beta-interferon. In some embodiments, the interferon isgamma-interferon. In some embodiments, the interferon isomega-interferon. In another embodiment, the interferon is a subspeciesof interferon such as a Type I or Type II interferon. In one embodiment,the subspecies of interferon (IFN) is IFN-α2a. In one embodiment, thesubspecies of interferon (IFN) is IFN-α2b. In one embodiment, thesubspecies of interferon (IFN) is IFN-β1a. In one embodiment, theinterferon (IFN) subspecies is IFN-β1b.

In some embodiments, GenBank Accession Nos. of non-limiting examples ofinterferons are listed in Table 1 below. In one embodiment, aninterferon of the present invention also refers to a homologue. In oneembodiment, the interferon amino acid sequence of the present inventionis at least 50% homologous to interferon sequences listed in Table 1 asdetermined using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters. In one embodiment, theinterferon amino acid sequence of the present invention is at least 60%homologous interferon sequences listed in Table 1 as determined usingBlastP software of the National Center of Biotechnology Information(NCBI) using default parameters. In one embodiment, the interferon aminoacid sequence of the present invention is at least 70% homologousinterferon sequences listed in Table 1 as determined using BlastPsoftware of the National Center of Biotechnology Information (NCBI)using default parameters. In one embodiment, the interferon amino acidsequence of the present invention is at least 80% homologous tointerferon sequences listed in Table 1 as determined using BlastPsoftware of the National Center of Biotechnology Information (NCBI)using default parameters. In one embodiment, the interferon amino acidsequence of the present invention is at least 90% homologous tointerferon sequences listed in Table 1 as determined using BlastPsoftware of the National Center of Biotechnology Information (NCBI)using default parameters. In one embodiment, the interferon amino acidsequence of the present invention is at least 95% homologous interferonsequences listed in Table 1 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters. In some embodiments, homology according to the presentinvention also encompasses deletions, insertions, or substitutionvariants, including an amino acid substitution thereof, and biologicallyactive polypeptide fragments thereof. In one embodiment, the cysteine inposition 17 of interferon 13 is substituted by a Serine (SEQ ID NO: 24).

Table 1 below lists examples of interferons with their respective NCBIsequence numbers.

TABLE 1 Interferon name NCBI sequence number interferon, α 1 NP_076918.1interferon, α 10 NP_002162.1 interferon, α 13 NP_008831.2 interferon, α14 NP_002163.1 interferon, α 16 NP_002164.1 interferon, α 17 NP_067091.1interferon, α 2 NP_000596.2 interferon, α 21 NP_002166.1 interferon, α 4NP_066546.1 interferon, α 5 NP_002160.1 interferon, α 6 NP_066282.1interferon, α 7 NP_066401.2 interferon, α 8 NP_002161.2 interferon, β 1NP_002167.1 interferon, ε1 NP_795372.1 interferon, γ NP_000610.2interferon, ε NP_064509.1 interferon, Ω1 NP_002168.1

In another embodiment, a method of treating or reducing a diseasetreatable or reducible by a cytokine or a pharmaceutical formulationcomprising the same, in a subject, comprises the step of administeringto a subject a therapeutically effective amount of the polypeptidecomprising a cytokine and CTP units as described herein, therebytreating or reducing a disease treatable or reducible by a cytokine in asubject.

In another embodiment, a method of treating or reducing a diseasetreatable or reducible by an interferon or a pharmaceutical formulationcomprising the same, in a subject, comprises the step of administeringto a subject a therapeutically effective amount of the polypeptidecomprising IFN protein and CTP units as described herein, therebytreating or reducing a disease treatable or reducible by an interferonin a subject.

In another embodiment, a disease treatable or reducible by an interferonis a Hepatitis C infection, cancer, bacterial infection, viralinfection, injury, multiple sclerosis, hairy cell leukemia, malignantmelanoma, Kaposi's sarcoma, bladder cancer, chronic myelocytic leukemia,kidney cancer, carcinoid tumors, non-Hodgkin's lymphoma, ovarian cancer,skin chronic Hepatitis C(CHC), condylomata accuminata (CA), chronicHepatitis B, follicular non-Hodgkin's lymphoma, chronic granulomatousdisease, Mycobacterium avium complex (MAC), pulmonary fibrosisosteoarthritis, and osteoporosis.

In another embodiment, polypeptides of the present invention comprisingIFN α-2a as well as pharmaceutical compositions comprising the same areindicated for hairy cell leukemia (HCL), acquired immune deficiencysyndrome(AIDS)-related Kaposi's sarcoma (KS), chronic-phase Philadelphia(Ph) chromosome-positive chronic myelogenous leukemia (CML) and chronicHepatitis C(CHC). IFN α-2a dosage varies depending on the indication. Inanother embodiment, the effectiveness of IFN α2a as an antineoplastic,immunomodulator and antiviral agent has been established.

In another embodiment, polypeptides of the present invention comprisingIFN α-2b as well as pharmaceutical compositions comprising the same areindicated for HCL, AIDS-related Kaposi's sarcoma and CHC. It is alsoindicated for condylomata accuminata (CA), chronic Hepatitis B,malignant melanoma and follicular non-Hodgkin's lymphoma. IFN α-2bdosage varies depending on its indication of usage.

In another embodiment, a polypeptide comprising an IFN protein, at leasta single CTP attached to its carboxy terminus, and at least a single CTPattached to its amino terminus is used to trigger an immune response. Inanother embodiment, a polypeptide comprising an IFN protein, a singleCTP attached to its amino terminus, and at least two CTP units attachedto its carboxy terminus is used to trigger an immune response. Inanother embodiment, a polypeptide comprising an IFN protein, a singleCTP attached to its amino terminus, and two CTP units attached to itscarboxy terminus is used to trigger an immune response. In anotherembodiment, a polypeptide comprising an IFN protein and CTP units isformulated in a pharmaceutical composition that is administered to asubject in need of triggering an immune response.

In another embodiment, a polypeptide comprising an IFN protein and CTPunits as described herein is used to trigger an immune response againsta viral infection. In another embodiment, a polypeptide comprising anIFN protein and CTP units is formulated in a pharmaceutical compositionthat is administered to a subject in need of triggering an immuneresponse against a viral infection.

In another embodiment, a polypeptide comprising an IFN β and CTP unitsas described herein is used to trigger an immune response via theenhancement of activity of lymphocyte cells. In another embodiment, apolypeptide comprising an IFN β and CTP units is formulated in apharmaceutical composition that is administered to a subject in need oftriggering an immune response via the enhancement of activity oflymphocyte cells.

In another embodiment, a polypeptide comprising a cytokine and CTP unitsas described herein is used as an anti-tumor agent. In anotherembodiment, a polypeptide comprising an IFN α and CTP units as describedherein is used as an anti-tumor agent. In another embodiment, apolypeptide comprising an IFN α and CTP units is formulated in apharmaceutical composition that is administered to a patient afflictedwith cancer.

In another embodiment, a polypeptide comprising an IFN protein and CTPunits as described herein is used equivalently to a regular or arecombinant interferon as known to one of average skill in the art. Inanother embodiment, a polypeptide comprising an IFN protein and CTPunits is formulated equivalently to a regular or a recombinantinterferon as known to one of average skill in the art.

In another embodiment, a polypeptide comprising a cytokine and CTP unitsas described herein modulates an immune response. In another embodiment,a polypeptide comprising a cytokine and CTP units as described hereinmodulates a cellular immune response. In another embodiment, apolypeptide comprising a cytokine and CTP units as described hereinmodulates an antibody immune response. In another embodiment, apolypeptide comprising a cytokine and CTP units as described hereininhibits an immune response as described herein. In another embodiment,a polypeptide comprising a cytokine and CTP units as described hereintrigger an immune response as described herein.

In another embodiment, a polypeptide comprising an IFN inhibits theactivity of T-cells, while simultaneously reducing the productioncytokines that operate in the inflammatory response to infection andinjury. In another embodiment, a polypeptide comprising an IFN proteinand CTP units as described herein enhances the activity of T-cells,while simultaneously reducing the production of cytokines that operatein the inflammatory response to infection and injury. In anotherembodiment, a polypeptide comprising an IFN protein and CTP units isformulated in a pharmaceutical composition that is administered to apatient in need of T-cell activity enhancement. In another embodiment, apolypeptide comprising an IFN protein and CTP units is formulated in apharmaceutical composition that is administered to a patient afflictedwith multiple sclerosis. In another embodiment, a polypeptide comprisingan IFN protein and CTP units is formulated in a pharmaceuticalcomposition that is administered to a patient afflicted with a HepatitisC infection.

In another embodiment, a cytokine is an interferon (IFN). In anotherembodiment, a cytokine is a type I interferon. In another embodiment,the interferon (IFN) is IFN-{tilde over (α)}. In another embodiment, theinterferon (IFN) is IFN-β. In another embodiment, the interferon (IFN)is IFN-γ. In another embodiment, an interferon (IFN) as described hereincomprises an amino acid sequence as described herein, including thesequences provided in FIG. 14. In another embodiment, a polypeptide ofthe invention comprising an interferon (IFN) peptide and at least oneCTP unit attached to an amino and/or a carboxy terminus of thepolypeptide as described herein comprises an amino acid sequence asdescribed herein, including the sequences provided in FIG. 14. Inanother embodiment, an interferon (IFN) peptide as described hereincomprises an amino acid sequence set forth in SEQ ID NO: 48. In anotherembodiment, SEQ ID NO: 48 comprises the following amino acid (AA)sequence:

MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGY LRN(SEQ ID NO: 48, Human Interferon-β 1a-MOD-9010).

In another embodiment, an interferon (IFN) peptide as described hereincomprises an amino acid sequence of human interferon β1a (hIFN β1a). Inanother embodiment, an interferon (IFN) peptide as described hereincomprises an amino acid sequence set fourth in GenBank Accession No.NP_(—)002167.1

In another embodiment, an interferon (IFN) as described herein isencoded by a nucleic acid sequence set forth in SEQ ID NO: 49. Inanother embodiment, SEQ ID NO: 49 comprises the following nucleic acid(NA) sequence: tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtgcttcagcaccaccgccctgagcatgagctacaacctgctgggcttcctgcagaggtccagcaacttccagtgccagaagctgctgtggcagctgaacggcaggctggaatactgcctgaaggacaggatgaacttcgacatcccagaggaaatcaagcagctgcagcagttccagaaggaggacgccgccctgaccatctacgagatgctgcagaacatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgagaccatcgtggagaacctgctggccaacgtgtaccaccagatcaaccacctgaaaaccgtgctggaagagaagctggaaaaggaggacttcaccaggggcaagctgatgagcagcctgcacctgaagaggtactacggcagaatcctgcactacctgaaggccaaggagtacagccactgcgcctggaccatcgtgagggtggagatcctgaggaacttctacttcatcaacaggctgaccggctacctgaggaactgatgagtccgcggccgc(SEQ ID NO: 49, Human Interferon-β1α-MOD-9010). In another embodiment,an interferon (IFN) peptide as described herein is encoded by a nucleicacid (NA) molecule of human interferon β1a (hIFN β1a). In anotherembodiment, an interferon (IFN) peptide as described herein is encodedby a nucleic acid (NA) molecule comprising a nucleic acid sequence setfourth in GenBank Accession No. NM 002176.

In another embodiment, an interferon (IFN) peptide as described hereincomprises an amino acid sequence set forth in SEQ ID NO: 50. In anotherembodiment, SEQ ID NO: 50 comprises the following amino acid (AA)sequence: TF*LQPFEAFALAQQVVGDTVRVVNMTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN (SEQ ID NO: 50).

In another embodiment, an interferon (IFN) peptide as described hereinis encoded by a nucleic acid sequence set forth in SEQ ID NO: 51. Inanother embodiment, SEQ ID NO: 51 comprises the following nucleic acid(NA) sequence:

(SEQ ID NO: 51) acattctaactgcaacctttcgaagcctttgctctggcacaacaggtagtaggcgacactgttcgtgttgtcaacatgaccaacaagtgtctcctccaaattgctctcctgttgtgcttctccactacagctctttccatgagctacaacttgcttggattcctacaaagaagcagcaattttcagtgtcagaagctcctgtggcaattgaatgggaggcttgaatactgcctcaaggacaggatgaactttgacatccctgaggagattaagcagctgcagcagttccagaaggaggacgccgcattgaccatctatgagatgctccagaacatctttgctattttcagacaagattcatctagcactggctggaatgagactattgttgagaacctcctggctaatgtctatcatcagataaaccatctgaagacagtcctggaagaaaaactggagaaagaagatttcaccaggggaaaactcatgagcagtctgcacctgaaaagatattatgggaggattctgcattacctgaaggccaaggagtacagtcactgtgcctggaccatagtcagagtggaaatcctaaggaacttttacttcattaacagacttacaggttacctccgaaactga.

In another embodiment, the cytokine as described herein comprises acytokine and at least three CTP units. In another embodiment, thepolypeptide as described herein comprises an interferon (IFN) peptideand three CTP units. In another embodiment, the cytokine as describedherein comprises an interferon (IFN) peptide-CTP polypeptide encoded byan amino acid sequence comprising the amino acid sequence set forth inSEQ ID NO: 52. In another embodiment, SEQ ID NO: 52 comprises thefollowing amino acid (AA) sequence:

(SEQ ID NO: 52, MOD-9011)MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNSSSSKAPPPSLPS PSRLPGPSDTPILPQ.

In another embodiment, the cytokine as described herein comprising aninterferon (IFN) peptide—and CTP is encoded by a nucleic acid moleculeset forth in SEQ ID NO: 53. In another embodiment, SEQ ID NO: 53comprises the following nucleic acid (NA) sequence:

(SEQ ID NO: 53, MOD-9011)tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtgcttcagcaccaccgccctgagcatgagctacaacctgctgggcttcctgcagaggtccagcaacttccagtgccagaagctgctgtggcagctgaacggcaggctggaatactgcctgaaggacaggatgaacttcgacatcccagaggaaatcaagcagctgcagcagttccagaaggaggacgccgccctgaccatctacgagatgctgcagaacatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgagaccatcgtggagaacctgctggccaacgtgtaccaccagatcaaccacctgaaaaccgtgctggaagagaagctggaaaaggaggacttcaccaggggcaagctgatgagcagcctgcacctgaagaggtactacggcagaatcctgcactacctgaaggccaaggagtacagccactgcgcctggaccatcgtgagggtggagatcctgaggaacttctacttcatcaacaggctgaccggctacctgaggaacagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgc cacagtgatga.

In another embodiment, the cytokine as described herein comprises aninterferon (IFN) peptide and two CTP units attached to its carboxyterminus. In another embodiment, the polypeptide as described hereincomprises an interferon (IFN) peptide-CTP (×2) encoded by an amino acidsequence comprising the amino acid sequence set forth in SEQ ID NO: 54.In another embodiment, SEQ ID NO: 54 comprises the following amino acid(AA) sequence:

(SEQ ID NO: 54, MOD-9012)MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ.

In another embodiment, the cytokine as described herein comprising aninterferon (IFN) peptide—and two CTP units attached to its carboxyterminus is encoded by a nucleic acid molecule set forth in SEQ ID NO:55. In another embodiment, SEQ ID NO: 55 comprises the following nucleicacid (NA) sequence:

(SEQ ID NO: 55, MOD-9012)tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtgcttcagcaccaccgccctgagcatgagctacaacctgctgggcttcctgcagaggtccagcaacttccagtgccagaagctgctgtggcagctgaacggcaggctggaatactgcctgaaggacaggatgaacttcgacatcccagaggaaatcaagcagctgcagcagttccagaaggaggacgccgccctgaccatctacgagatgctgcagaacatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgagaccatcgtggagaacctgctggccaacgtgtaccaccagatcaaccacctgaaaaccgtgctggaagagaagctggaaaaggaggacttcaccaggggcaagctgatgagcagcctgcacctgaagaggtactacggcagaatcctgcactacctgaaggccaaggagtacagccactgcgcctggaccatcgtgagggtggagatcctgaggaacttctacttcatcaacaggctgaccggctacctgaggaacagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgacacaccaatcctgccacagagcagctcctctaaggcccctcctccatccctgccatccccctcccggctgcctggcccctctgacacccctatcctgcctcagtgatgaaggtc tggatccgcggccgc.

In another embodiment, the cytokine as described herein comprises aninterferon

(IFN) peptide, a single CTP unit attached to the IFN's amino terminus,and two CTP units attached to the IFN's carboxy terminus. In anotherembodiment, the polypeptide as described herein comprises an interferon(IFN) peptide, a single CTP unit attached to the IFN's amino terminusand two CTP units attached in tandem to the IFN's carboxy terminus. Inanother embodiment, the polypeptide as described herein comprises (fromamino to carboxy termini): CTP (×1)-interferon (IFN) peptide—CTP (×2)comprising an amino acid sequence set forth in SEQ ID NO: 56. In anotherembodiment, SEQ ID NO: 56 comprises the following amino acid (AA)sequence:

(SEQ ID NO: 56, MOD-9013)MTNKCLLQIALLLCFSTTALSSSSSKAPPPSLPSPSRLPGPSDTPILPQMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ.

In another embodiment, the cytokine as described herein comprising aninterferon (IFN) peptide, a single CTP unit attached to the IFN's aminoterminus and two CTP units attached to the IFN's carboxy terminus isencoded by a nucleic acid molecule set forth in SEQ ID NO: 57. Inanother embodiment, SEQ ID NO: 57 comprises the following nucleic acid(NA) sequence:

(SEQ ID NO: 57, MOD-9013)tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtgcttcagcaccaccgccctgagcagcagcagctccaaggccccaccccccagcctgcccagccccagcagactgccaggccccagcgacacccccatcctgccccagatgagctacaacctgctgggcttcctgcagaggtccagcaacttccagtgccagaagctgctgtggcagctgaacggcaggctggaatactgcctgaaggacaggatgaacttcgacatcccagaggaaatcaagcagctgcagcagttccagaaggaggacgccgccctgaccatctacgagatgctgcagaacatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgagaccatcgtggagaacctgctggccaacgtgtaccaccagatcaaccacctgaaaaccgtgctggaagagaagctggaaaaggaggacttcaccaggggcaagctgatgagcagcctgcacctgaagaggtactacggcagaatcctgcactacctgaaggccaaggagtacagccactgcgcctggaccatcgtgagggtggagatcctgaggaacttctacttcatcaacaggctgaccggctacctgaggaacagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgacacaccaatcctgccacagagcagctcctctaaggcccctcctccatccctgccatccccctcccggctgcctggcccctctgacacccctatcctgcctcagtgatgaaggtctggatccgcggccgc.

In another embodiment, the cytokine as described herein comprises aninterferon (IFN) peptide, a single CTP attached to the IFN's aminoterminus, and a single CTP located within an IFN coding sequence. Inanother embodiment, the polypeptide as described herein comprises (fromamino to carboxy termini): CTP (×1)-interferon (IFN) peptide (fragment1)—CTP-interferon (IFN) peptide (fragment 2) comprising an amino acidsequence set forth in SEQ ID NO: 58. In another embodiment, SEQ ID NO:58 comprises the following amino acid (AA) sequence:

(SEQ ID NO: 58, MOD-9014)MTNKCLLQIALLLCFSTTALSSSSSKAPPPSLPSPSRLPGPSDTPILPQMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNSSSSKAPPPSLPSPSRLPGPSDTPILPQMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFI NRLTGYLRN.

In another embodiment, the cytokine as described herein comprising aninterferon (IFN) peptide, a single CTP unit attached to the IFN's aminoterminus, and a single CTP unit located within the IFN coding sequenceis encoded by a nucleic acid molecule set forth in SEQ ID NO: 59. Inanother embodiment, SEQ ID NO: 59 comprises the following nucleic acid(NA) sequence:

(SEQ ID NO: 59, MOD-9014)tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtgcttcagcaccaccgccctgagcagcagcagctccaaggccccaccccccagcctgcccagccccagcaggctgccaggccccagcgacacccccatcctgccccagatgagctacaacctgctgggcttcctgcagaggtccagcaacttccagtgccagaaactgctgtggcagctgaacggcaggctggaatactgcctgaaggaccggatgaacttcgacatccccgaagagatcaagcagctgcagcagttccagaaagaggacgccgccctgaccatctacgagatgctgcagaacatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgagaccatcgtggagaacctgctggccaacgtgtaccaccagatcaaccacctgaaaaccgtgctggaagagaagctggaaaaagaggacttcaccaggggcaagctgatgagcagcctgcacctgaagaggtactacggcagaatcctgcactacctgaaggccaaagagtacagccactgcgcctggaccatcgtgagggtggagatcctgcggaacttctacttcatcaacaggctgaccggctacctgaggaacagctccagcagcaaggcccctccaccctccctgccctccccaagcagactgcccggaccctccgacacaccaattctgccacagatgtcctacaatctgctcggatttctgcagcgctcctccaactttcagtgtcagaagctcctctggcagctcaatggccgcctggaatattgtctgaaagacagaatgaattttgacatcccagaggaaattaaacagctccagcagtttcagaaagaagatgctgctctcacaatctatgaaatgctccagaatatctttgcaatctttcgccaggacagctcctccaccgggtggaatgagacaattgtcgagaatctgctcgccaatgtctatcatcagatcaatcacctcaagacagtcctcgaagaaaaactcgaaaaagaagatttcacacgcggcaaactgatgtcctccctgcatctgaagcgctactatgggcgcatcctgcattatctgaaagctaaagaatactcccactgtgcttggacaattgtgcgcgtcgagatcctgagaaacttttatttcattaaccgcctgacaggatacctgcgcaactgatgaaggtctg gatgcggccgc.

In another embodiment, the cytokine as described herein comprises aninterferon (IFN) peptide and a single CTP unit attached to its aminoterminus. In another embodiment, the polypeptide as described hereincomprises an interferon (IFN) peptide-CTP comprising an amino acidsequence set forth in SEQ ID NO: 60. In another embodiment, SEQ ID NO:60 comprises the following amino acid (AA) sequence:

(SEQ ID NO: 60, MOD-9015)MTNKCLLQIALLLCFSTTALSSSSSKAPPPSLPSPSRLPGPSDTPILPQMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEIL RNFYFINRLTGYLRN*.

In another embodiment, the polypeptide as described herein comprising aninterferon (IFN) peptide—and a single CTP attached to its amino terminusis encoded by a nucleic acid molecule set forth in SEQ ID NO: 61. Inanother embodiment, SEQ ID NO: 61 comprises the following nucleic acid(NA) sequence:

(SEQ ID NO: 61, MOD-9015)tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtgcttcagcaccaccgccctgagcagcagcagctccaaggccccaccccccagcctgcccagccccagcaggctgccaggccccagcgacacccccatcctgccccagatgagctacaacctgctgggcttcctgcagaggtccagcaacttccagtgccagaaactgctgtggcagctgaacggcaggctggaatactgcctgaaggaccggatgaacttcgacatccccgaagagatcaagcagctgcagcagttccagaaagaggacgccgccctgaccatctacgagatgctgcagaacatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgagaccatcgtggagaacctgctggccaacgtgtaccaccagatcaaccacctgaaaaccgtgctggaagagaagctggaaaaagaggacttcaccaggggcaagctgatgagcagcctgcacctgaagaggtactacggcagaatcctgcactacctgaaggccaaagagtacagccactgcgcctggaccatcgtgagggtggagatcctgcggaacttctacttcatcaacaggctgaccggctacctgaggaactgatgagtccgcggccgc.

In another embodiment, the polypeptide as described herein comprises aninterferon (IFN) peptide, a single CTP unit attached to its aminoterminus, and a single CTP unit attached to its carboxy terminus. Inanother embodiment, the polypeptide as described herein comprises aninterferon (IFN) peptide-CTP comprising an amino acid sequence set forthin SEQ ID NO: 62. In another embodiment, SEQ ID NO: 62 comprises thefollowing amino acid (AA) sequence:

(SEQ ID NO: 62, MOD-9016)MTNKCLLQIALLLCFSTTALSSSSSKAPPPSLPSPSRLPGPSDTPILPQMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRNSSSSKAPPPSLPSPSRLPGPSDTPILPQ*.

In another embodiment, the cytokine as described herein comprising aninterferon (IFN) peptide, a single CTP unit attached to its aminoterminus, and a single CTP unit attached to its carboxy terminus isencoded by a nucleic acid molecule set forth in SEQ ID NO: 63. Inanother embodiment, SEQ ID NO: 63 comprises the following nucleic acid(NA) sequence:

(SEQ ID NO: 63, MOD-9016)tctagaggacatgaccaacaagtgcctgctgcagatcgccctgctgctgtgcttcagcaccaccgccctgagcagcagcagctccaaggccccaccccccagcctgcccagccccagcagactgccaggccccagcgacacccccatcctgccccagatgagctacaacctgctgggcttcctgcagaggtccagcaacttccagtgccagaagctgctgtggcagctgaacggcaggctggaatactgcctgaaggacaggatgaacttcgacatcccagaggaaatcaagcagctgcagcagttccagaaggaggacgccgccctgaccatctacgagatgctgcagaacatcttcgccatcttcaggcaggacagcagcagcaccggctggaacgagaccatcgtggagaacctgctggccaacgtgtaccaccagatcaaccacctgaaaaccgtgctggaagagaagctggaaaaggaggacttcaccaggggcaagctgatgagcagcctgcacctgaagaggtactacggcagaatcctgcactacctgaaggccaaggagtacagccactgcgcctggaccatcgtgagggtggagatcctgaggaacttctacttcatcaacaggctgaccggctacctgaggaacagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagtgatgaaggtc tggatgcggccgc.

In another embodiment, an interferon β peptide comprises SEQ ID NO: 6comprising the following amino acid (AA) sequence:

(SEQ ID NO: 68) MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEI LRNFYFINRLTGYLRN.

In another embodiment, the methods of the present invention provide aninterferon beta 1 peptide having additionally one CTP amino acid peptideon the N-terminus and two CTP amino acid peptides on the C-terminus fortreating or inhibiting multiple sclerosis. In another embodiment, themethods of the present invention provide an interferon beta 1 proteincomprising an interferon beta 1 sequence as provided herein for treatingdiseases such as, but not limited to, multiple sclerosis, cancer, orviral infections. In another embodiment, the methods of the presentinvention provide an interferon beta 1 protein comprising an interferonbeta 1 sequence as provided herein for treating diseases such as, butnot limited to, multiple sclerosis, cancer, or viral infections. Inanother embodiment, the methods of the present invention provide aninterferon beta 1 peptide set forth in SEQ ID NO: 56 for treatingdiseases such as, but not limited to, multiple sclerosis, cancer, orviral infections. In another embodiment, the methods of the presentinvention provide an interferon beta 1 peptide set forth in SEQ ID NO:58 for treating diseases such as, but not limited to, multiplesclerosis, cancer, or viral infections. In another embodiment, themethods of the present invention provide an interferon beta 1 peptideset forth in SEQ ID NO: 60 for treating diseases such as, but notlimited to, multiple sclerosis, cancer, or viral infections. In anotherembodiment, the methods of the present invention provide an interferonbeta 1 peptide set forth in SEQ ID NO: 62 for treating diseases such as,but not limited to, multiple sclerosis, cancer, or viral infections.

As provided herein, attachment of a CTP sequence to both the amino andcarboxy termini of the EPO protein results in increased potency atstimulating erythropoiesis (FIGS. 3-5 and Table 6 of Example 4), ascompared to recombinant EPO and other combinations of EPO and CTP. Insome embodiments, an EPO attached to three CTP sequences does not impairbinding to its receptor as evidenced in Table 4 of Example 3, whichdemonstrates that EPO attached to three CTP sequences is equallyeffective at stimulating proliferation of TF-1 cells as wild-type EPO.

In some embodiments, “homology” according to the present invention alsoencompasses deletions, insertions, or substitution variants, includingan amino acid substitution thereof, and biologically active polypeptidefragments thereof. In one embodiment, the substitution variant comprisesa substitution of the glycine in position 104 of erythropoietin aminoacid sequence with a serine (SEQ ID NO: 22).

In another embodiment, the methods of the present invention provide anEPO protein having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO protein having additionally one CTPamino acid peptide on the N-terminus and two CTP amino acid peptides onthe C-terminus for the treatment of anemia.

In another embodiment, the methods of the present invention provide anEPO protein as set forth in SEQ ID NO: 1 having additionally at leastone CTP amino acid peptide on the N-terminus for the treatment ofanemia. In another embodiment, the methods of the present inventionprovide an EPO protein as set forth in SEQ ID NO: 1 having additionallyat least one CTP amino acid peptide on the N-terminus and at least oneadditional CTP amino acid peptide on the C-terminus for the treatment ofanemia. In another embodiment, the methods of the present inventionprovide an EPO protein as set forth in SEQ ID NO: 2 having additionallyat least one CTP amino acid peptide on the N-terminus and at least oneCTP amino acid peptide on the C-terminus for the treatment of anemia. Inanother embodiment, the methods of the present invention provide an EPOprotein as set forth in SEQ ID NO: 3 having additionally at least oneCTP amino acid peptide on the N-terminus and at least one CTP amino acidpeptide on the C-terminus for the treatment of anemia. In anotherembodiment, the methods of the present invention provide an EPO proteinas set forth in SEQ ID NO: 4 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of anemia. In another embodiment,the methods of the present invention provide an EPO protein as set forthin SEQ ID NO: 5 having additionally at least one CTP amino acid peptideon the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of anemia. In another embodiment, themethods of the present invention provide an EPO protein as set forth inSEQ ID NO: 6 having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO protein as set forth in SEQ ID NO: 16having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia. In another embodiment, the methods of thepresent invention provide an EPO protein as set forth in SEQ ID NO: 22having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of anemia.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO protein having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for the treatment of anemia. Inanother embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO protein having additionally oneCTP amino acid peptide on the N-terminus and two CTP amino acid peptideson the C-terminus for the treatment of anemia. In another embodiment,the methods of the present invention provide a nucleic acid sequence asset forth in SEQ ID NO: 20 encoding an EPO protein and one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for the treatment of anemia. In another embodiment,the methods of the present invention provide a nucleic acid sequence asset forth in SEQ ID NO: 21 encoding an EPO protein and one CTP aminoacid peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for the treatment of anemia.

In another embodiment, the methods of the present invention provide anEPO protein having additionally one CTP amino acid peptide on theN-terminus and two CTP amino acid peptides on the C-terminus forinhibiting anemia, for treating or inhibiting tumor-associated anemiafor treating or inhibiting tumor hypoxia for treating or inhibitingchronic infections such as HIV, inflammatory bowel disease, or septicepisodes, for treating fatigue syndrome following cancer chemotherapy,for improving stem cell engraftment, or for increasing the survival rateof a patient with aplastic anemia or myelodysplastic syndrome. Inanother embodiment, the methods of the present invention provide an EPOprotein as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 16 or SEQ ID NO: 22 havingadditionally one CTP peptide on the N-terminus and two CTP peptides onthe C-terminus for inhibiting anemia, for treating or inhibitingtumor-associated anemia for treating or inhibiting tumor hypoxia fortreating or inhibiting chronic infections such as HIV, inflammatorybowel disease, or septic episodes, for treating fatigue syndromefollowing cancer chemotherapy, for improving stem cell engraftment, orfor increasing the survival rate of a patient with aplastic anemia ormyelodysplastic syndrome.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an EPO protein having additionally oneCTP peptide on the N-terminus and two CTP amino acid peptides on theC-terminus for inhibiting anemia, for treating or inhibitingtumor-associated anemia for treating or inhibiting tumor hypoxia fortreating or inhibiting chronic infections such as HIV, inflammatorybowel disease, or septic episodes, for treating fatigue syndromefollowing cancer chemotherapy, for improving stem cell engraftment, orfor increasing the survival rate of a patient with aplastic anemia ormyelodysplastic syndrome. In another embodiment, the methods of thepresent invention provide a nucleic acid sequence as set forth in SEQ IDNO: 20, SEQ ID NO: 21 encoding an EPO protein and one CTP peptide on theN-terminus and two CTP peptides on the C-terminus for inhibiting anemia,for treating or inhibiting tumor-associated anemia, for treating orinhibiting tumor hypoxia, for treating or inhibiting chronic infectionssuch as HIV, inflammatory bowel disease, or septic episodes, fortreating fatigue syndrome following cancer chemotherapy, for improvingstem cell engraftment, or for increasing the survival rate of a patientwith aplastic anemia or myelodysplastic syndrome.

In another embodiment, human growth hormone (hGH) is the cytokine asdescribed herein. In another embodiment, CTP-hGH-CTP-CTP constructs ofthe invention bind adipocytes and stimulate them to break downtriglyceride and suppresses their ability to take up and accumulatecirculating lipids. In another embodiment, CTP-hGH-CTP-CTP constructs ofthe invention exert indirect effects mediated primarily by aninsulin-like growth factor-I (IGF-I) (as shown in the examples section).

In another embodiment, CTP-hGH-CTP-CTP constructs of the inventionstimulate body growth by stimulating the liver and other tissues tosecrete IGF-I. In another embodiment, IGF-I stimulates proliferation ofchondrocytes, resulting in bone growth.

In another embodiment, CTP-hGH-CTP-CTP constructs of the inventioninduce metabolic effects on protein, lipid and carbohydrate metabolism.In another embodiment, CTP-hGH-CTP-CTP constructs of the invention havea direct effect. In another embodiment, CTP-hGH-CTP-CTP constructs ofthe invention have an indirect effect through induction of IGF-I. Inanother embodiment, CTP-hGH-CTP-CTP constructs include constructscomprising a leader peptide. In another embodiment, CTP-hGH-CTP-CTPconstructs include truncated constructs.

In another embodiment, CTP-hGH-CTP-CTP constructs of the inventionstimulate protein anabolism in a tissue. In another embodiment,CTP-hGH-CTP-CTP constructs of the invention stimulate amino acid uptake,increased protein synthesis, and decreased oxidation of proteins.

In another embodiment, CTP-hGH-CTP-CTP constructs of the inventionstimulate fat metabolism. In another embodiment, CTP-hGH-CTP-CTPconstructs of the invention stimulate the utilization of fat bystimulating triglyceride breakdown and oxidation in adipocytes.

In another embodiment, CTP-hGH-CTP-CTP constructs of the inventionstimulate carbohydrate metabolism. In another embodiment,CTP-hGH-CTP-CTP constructs of the invention maintain blood glucosewithin a normal range. In another embodiment, CTP-hGH-CTP-CTP constructsof the invention comprise an anti-insulin activity. In anotherembodiment, CTP-hGH-CTP-CTP constructs of the invention suppress theabilities of insulin to stimulate uptake of glucose in peripheraltissues and enhance glucose synthesis in the liver. In anotherembodiment, CTP-hGH-CTP-CTP constructs of the invention stimulateinsulin secretion, leading to hyperinsulinemia.

In another embodiment, CTP-hGH-CTP-CTP constructs of the invention areused to compensate for limited or no production of growth hormone in asubject. In another embodiment, CTP-hGH-CTP-CTP constructs of theinvention compensate for limited or no production of growthhormone-releasing hormone (GHRH). In another embodiment, CTP-hGH-CTP-CTPconstructs of the invention compensate for the increased activity ofsomatostatin. In another embodiment, CTP-hGH-CTP-CTP constructs of theinvention compensate for limited or no production of ghrelin.

In another embodiment, CTP-hGH-CTP-CTP constructs of the invention areused to treat diseases associated with lesions in either thehypothalamus, the pituitary, or in target cells. In another embodiment,CTP-hGH constructs of the invention are used to treat diseasesassociated with reduced target cell's response to the hormone.

In another embodiment, CTP-hGH-CTP-CTP constructs of the invention areused to treat children with severe growth retardation. In anotherembodiment, CTP-hGH-CTP-CTP constructs of the invention are used totreat children of pathologically short stature. In another embodiment,CTP-hGH-CTP-CTP constructs of the invention are used to enhance athleticperformance. In another embodiment, CTP-hGH-CTP-CTP constructs of theinvention are used to treat symptoms of aging. In another embodiment,CTP-hGH constructs of the invention are used to treat cosmetic symptomsof aging.

In another embodiment, CTP-hGH-CTP-CTP constructs of the invention areused for enhancing milk production in a female subject. In anotherembodiment, CTP-cowGH-CTP-CTP constructs of the invention are used forenhancing milk production in dairy cattle. In another embodiment,CTP-animal-GH-CTP-CTP constructs of the invention are used in animalagriculture technology. In another embodiment, CTP-farmanimal-GH-CTP-CTP constructs of the invention are used for enhancinggrowth of farm animal such as, but not limited to, pigs.

In some embodiments, human growth hormone (hGH) is utilized according tothe teachings of the present invention. In some embodiments, theattachment of CTP sequences to both the amino and carboxy termini of thehGH protein results in increased potency (FIGS. 11). In someembodiments, the attachment of CTP sequences to both the amino andcarboxy termini of the hGH protein results in prolonged in vivoactivity. In one embodiment, CTP-hGH polypeptides of the presentinvention are set forth in SEQ ID NOs: 39-41.

In one embodiment, the phrase “human growth hormone” (hGH) refers to apolypeptide, such as that set forth in Genbank Accession No. P01241 (SEQID NO: 47), exhibiting hGH activity (i.e. stimulation of growth).

In one embodiment, “human growth hormone” (hGH) refers to a polypeptide,such as that set forth in Genbank Accession No. P01241, exhibiting hGHactivity (i.e. stimulation of growth). In one embodiment, an hGH of thepresent invention also refers to homologues. In one embodiment, an hGHamino acid sequence of the present invention is at least 50% homologousto an hGH sequence set forth in GenBank Accession No. P01241 asdetermined using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters. In one embodiment, an hGHamino acid sequence of the present invention is at least 60% homologousto an hGH sequence set forth in GenBank Accession No. P01241 asdetermined using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters. In one embodiment, an hGHamino acid sequence of the present invention is at least 70% homologousto an hGH sequence set forth in GenBank Accession No. P01241 asdetermined using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters. In one embodiment, an hGHamino acid sequence of the present invention is at least 80% homologousto an hGH sequence set forth in GenBank Accession No. P01241 asdetermined using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters. In one embodiment, an hGHamino acid sequence of the present invention is at least 90% homologousto an hGH sequence set forth in GenBank Accession No. P01241 asdetermined using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters. In one embodiment, an hGHamino acid sequence of the present invention is at least 95% homologousto an hGH sequence set forth in GenBank Accession No. P01241 asdetermined using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters.

Exemplary CTP-hGH polypeptides of the present invention are set forth inSEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41.

In another embodiment, the methods of the present invention provide aCTP-hGH-CTP-CTP construct used for stimulating muscle growth, increasingcardiac function, stimulating bone growth, maintaining muscle integrity,balancing muscle metabolism, inducing muscle buildup, inducing de-novomuscle build-up, enhancing bone load, treating symptoms associated withosteoporosis, treating a wasting disease, increasing lipolysis,improving fluid balance, treating osteoporosis, improving lung function,improving immunity, regrowing a vital organ, increasing sense ofwell-being, restoring REM sleep, or any combination thereof. In anotherembodiment, the methods of the present invention provide an hGH proteinas set forth in SEQ ID NO: 23 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for stimulating muscle growth, increasing cardiacfunction, stimulating bone growth, maintaining muscle integrity,balancing muscle metabolism, inducing muscle buildup, inducing de-novomuscle build-up, enhancing bone load, treating symptoms associated withosteoporosis, treating a wasting disease, increasing lipolysis,improving fluid balance, treating osteoporosis, improving lung function,improving immunity, regrowing a vital organ, increasing sense ofwell-being, restoring REM sleep, or any combination thereof. In anotherembodiment, the methods of the present invention provide an hGH proteinas set forth in SEQ ID NO: 36 having additionally at least one CTP aminoacid peptide on the N-terminus and at least one CTP amino acid peptideon the C-terminus for stimulating muscle growth, increasing cardiacfunction, stimulating bone growth, maintaining muscle integrity,balancing muscle metabolism, inducing muscle buildup, inducing de-novomuscle build-up, enhancing bone load, treating symptoms associated withosteoporosis, treating a wasting disease, increasing lipolysis,improving fluid balance, treating osteoporosis, improving lung function,improving immunity, regrowing a vital organ, increasing sense ofwell-being, restoring REM sleep, or any combination thereof. In anotherembodiment, the methods of the present invention provide an hGH proteinas set forth in SEQ ID NO: 37 having additionally at least one CTP aminoacid peptide on the N-terminus for stimulating muscle growth, increasingcardiac function, stimulating bone growth, maintaining muscle integrity,balancing muscle metabolism, inducing muscle buildup, inducing de-novomuscle build-up, enhancing bone load, treating symptoms associated withosteoporosis, treating a wasting disease, increasing lipolysis,improving fluid balance, treating osteoporosis, improving lung function,improving immunity, regrowing a vital organ, increasing sense ofwell-being, restoring REM sleep, or any combination thereof. In anotherembodiment, the methods of the present invention provide an hGH proteinas set forth in SEQ ID NO: 38 having additionally at least one CTP aminoacid peptide on the N-terminus for stimulating muscle growth, increasingcardiac function, stimulating bone growth, maintaining muscle integrity,balancing muscle metabolism, inducing muscle buildup, inducing de-novomuscle build-up, enhancing bone load, treating symptoms associated withosteoporosis, treating a wasting disease, increasing lipolysis,improving fluid balance, treating osteoporosis, improving lung function,improving immunity, regrowing a vital organ, increasing sense ofwell-being, restoring REM sleep, or any combination thereof. In anotherembodiment, the methods of the present invention provide an hGH proteinas set forth in SEQ ID NO: 39 for stimulating muscle growth, increasingcardiac function, stimulating bone growth, maintaining muscle integrity,balancing muscle metabolism, inducing muscle buildup, inducing de-novomuscle build-up, enhancing bone load, treating symptoms associated withosteoporosis, treating a wasting disease, increasing lipolysis,improving fluid balance, treating osteoporosis, improving lung function,improving immunity, regrowing a vital organ, increasing sense ofwell-being, restoring REM sleep, or any combination thereof. In anotherembodiment, the methods of the present invention provide an hGH proteinas set forth in SEQ ID NO: 40 for stimulating muscle growth, increasingcardiac function, stimulating bone growth, maintaining muscle integrity,balancing muscle metabolism, inducing muscle buildup, inducing de-novomuscle build-up, enhancing bone load, treating symptoms associated withosteoporosis, treating a wasting disease, increasing lipolysis,improving fluid balance, treating osteoporosis, improving lung function,improving immunity, regrowing a vital organ, increasing sense ofwell-being, restoring REM sleep, or any combination thereof. In anotherembodiment, the methods of the present invention provide an hGH proteinas set forth in SEQ ID NO: 41 for stimulating muscle growth, increasingcardiac function, stimulating bone growth, maintaining muscle integrity,balancing muscle metabolism, inducing muscle buildup, inducing de-novomuscle build-up, enhancing bone load, treating symptoms associated withosteoporosis, treating a wasting disease, increasing lipolysis,improving fluid balance, treating osteoporosis, improving lung function,improving immunity, regrowing a vital organ, increasing sense ofwell-being, restoring REM sleep, or any combination thereof. In anotherembodiment, the methods of the present invention provide an hGH proteinas set forth in SEQ ID NO: 42 having additionally at least one CTP aminoacid peptide on the N-terminus for stimulating muscle growth, increasingcardiac function, stimulating bone growth, maintaining muscle integrity,balancing muscle metabolism, inducing muscle buildup, inducing de-novomuscle build-up, enhancing bone load, treating symptoms associated withosteoporosis, treating a wasting disease, increasing lipolysis,improving fluid balance, treating osteoporosis, improving lung function,improving immunity, regrowing a vital organ, increasing sense ofwell-being, restoring REM sleep, or any combination thereof. In anotherembodiment, the methods of the present invention provide an hGH proteinmodified by CTPs as described herein for stimulating muscle growth,increasing cardiac function, stimulating bone growth, maintaining muscleintegrity, balancing muscle metabolism, inducing muscle buildup,inducing de-novo muscle build-up, enhancing bone load, treating symptomsassociated with osteoporosis, treating a wasting disease, increasinglipolysis, improving fluid balance, treating osteoporosis, improvinglung function, improving immunity, regrowing a vital organ, increasingsense of well-being, restoring REM sleep, or any combination thereof.

In another embodiment, the methods of the present invention provide anucleic acid sequence encoding an hGH protein as described herein. Inanother embodiment, the methods of the present invention provides anucleic acid of SEQ ID NO: 45 encoding an hGH protein comprising one CTPamino acid peptide on the N-terminus and two CTP amino acid peptides onthe C-terminus for stimulating muscle growth, increasing cardiacfunction, stimulating bone growth, maintaining muscle integrity,balancing muscle metabolism, inducing muscle buildup, inducing de-novomuscle build-up, enhancing bone load, treating symptoms associated withosteoporosis, treating a wasting disease, increasing lipolysis,improving fluid balance, treating osteoporosis, improving lung function,improving immunity, regrowing a vital organ, increasing sense ofwell-being, restoring REM sleep, or any combination thereof. In anotherembodiment, the methods of the present invention provide a nucleic acidof SEQ ID NO: 46 encoding an hGH protein and one CTP amino acid peptideon the N-terminus and two CTP amino acid peptides on the C-terminus forstimulating muscle growth, increasing cardiac function, stimulating bonegrowth, maintaining muscle integrity, balancing muscle metabolism,inducing muscle buildup, inducing de-novo muscle build-up, enhancingbone load, treating symptoms associated with osteoporosis, treating awasting disease, increasing lipolysis, improving fluid balance, treatingosteoporosis, improving lung function, improving immunity, regrowing avital organ, increasing sense of well-being, restoring REM sleep, or anycombination thereof.

In some embodiments, glucagon-like peptide-1 is utilized according tothe teachings of the present invention. In some embodiments, theattachment of CTP sequences to both the amino and carboxy termini of a“glucagon-like peptide-1” results in increased potency. In someembodiments, the attachment of CTP to both the amino and carboxy terminiof a peptide results in prolonged in vivo activity. In some embodiments,the attachment of CTP to both the amino and carboxy termini of theglucagon-like peptide-results in prolonged in vivo activity.

In one embodiment, “glucagon-like peptide-1” (GLP-1) refers to amammalian polypeptide. In one embodiment, “glucagon-like peptide-1”(GLP-1) refers to a human polypeptide. In some embodiments, GLP-1 iscleaved from the glucagon preproprotein (Genbank ID No. NP002045) thathas the ability to bind to the GLP-1 receptor and initiate a signaltransduction pathway resulting in insulinotropic activity. In oneembodiment, “insulinotropic activity” refers to the ability to stimulateinsulin secretion in response to elevated glucose levels, therebycausing glucose uptake by cells and decreased plasma glucose levels. Insome embodiments, GLP-1 polypeptides include, but are not limited tothose described in U.S. Pat. No. 5,118,666; which is incorporated byreference herein.

In one embodiment, “GLP-1” refers to a polypeptide, such as that setforth in SEQ ID NO: 25 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters. In one embodiment, a GLP-1 of the present invention alsorefers to a GLP-1 homologue. In one embodiment, a GLP-1 amino acidsequence of the present invention is at least 50% homologous to GLP-1sequences set forth in SEQ ID NO: 25 as determined using BlastP softwareof the National Center of Biotechnology Information (NCBI) using defaultparameters. In one embodiment, a GLP-1 amino acid sequence of thepresent invention is at least 60% homologous to GLP-1 sequences setforth in SEQ ID NO: 25 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters. In one embodiment, a GLP-1 amino acid sequence of thepresent invention is at least 70% homologous to GLP-1 sequences setforth in SEQ ID NO: 25 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters. In one embodiment, a GLP-1 amino acid sequence of thepresent invention is at least 80% homologous to GLP-1 sequences setforth in SEQ ID NO: 25 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters. In one embodiment, a GLP-1 amino acid sequence of thepresent invention is at least 90% homologous to GLP-1 sequences setforth in SEQ ID NO: 25 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters. In one embodiment, a GLP-1 amino acid sequence of thepresent invention is at least 95% homologous to GLP-1 sequences setforth in SEQ ID NO: 25 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters.

In another embodiment, the methods of the present invention provides aGLP-1 peptide having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor treating or inhibiting type II diabetes. In another embodiment, themethods of the present invention provides a GLP-1 peptide havingadditionally one CTP amino acid peptide on the N-terminus and two CTPamino acid peptides on the C-terminus for treating or inhibiting type IIdiabetes. In another embodiment, the methods of the present inventionprovides a GLP-1 peptide set forth in SEQ ID NO: 25 having additionallyat least one CTP amino acid peptide on the N-terminus and at least oneCTP amino acid peptide on the C-terminus for treating or inhibiting typeII diabetes.

In another embodiment, the polypeptide sequence-of-interest is aninsulin-like growth factor. In another embodiment, the polypeptidesequence-of-interest is an epidermal growth factor. In anotherembodiment, the polypeptide sequence-of-interest is an acidic or basicfibroblast growth factor. In another embodiment, the polypeptidesequence-of-interest is a platelet-derived growth factor. In anotherembodiment, the polypeptide sequence-of-interest is a granulocyte-CSF.In another embodiment, the polypeptide sequence-of-interest is amacrophage-CSF. In another embodiment, the polypeptidesequence-of-interest is an IL-2. In another embodiment, the polypeptidesequence-of-interest is an IL-3. In another embodiment, the polypeptidesequence-of-interest is a tumor necrosis factor. In another embodiment,the polypeptide sequence-of-interest is an LHRH. In another embodiment,the polypeptide sequence-of-interest is an LHRH analog. In anotherembodiment, the polypeptide sequence-of-interest is a somatostatin. Inanother embodiment, the polypeptide sequence-of-interest is a growthhormone releasing factor. In another embodiment, the polypeptidesequence-of-interest is an endorphin. In another embodiment, thepolypeptide sequence-of-interest is an alveolar surfactant protein. Inanother embodiment, the polypeptide sequence-of-interest is anatriuretic factor. In another embodiment, the polypeptidesequence-of-interest is an adhesion.

In another embodiment, the polypeptide sequence-of-interest is anangiostatin. In another embodiment, the polypeptide sequence-of-interestis an endostatin. In another embodiment, the polypeptidesequence-of-interest is a receptor peptide. In another embodiment, thepolypeptide sequence-of-interest is a receptor binding ligand. Inanother embodiment, the polypeptide sequence-of-interest is an antibody.In another embodiment, the polypeptide sequence-of-interest is anantibody fragment. In another embodiment, the polypeptidesequence-of-interest is a peptide or a protein including any modifiedform. In another embodiment, the polypeptide of the invention comprisesa cytokine having additionally at least one CTP amino acid peptide onthe N-terminus and one CTP amino acid peptide on the C-terminus. Inanother embodiment, the cytokine having additionally at least one CTPamino acid peptide on the N-terminus and two CTP amino acid peptides onthe C-terminus is selected from lymphokines, monokines, chemokine, andinterleukin. In another embodiment, the cytokine comprises an autocrineaction activity. In another embodiment, the cytokine comprises aparacrine action activity. In another embodiment, the cytokine comprisesan endocrine action activity.

In another embodiment, the methods of the present invention provideinsulin having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of diabetes.

In another embodiment, the methods of the present invention providealbumin having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of hypovolemic shock, hemodialysis or cardiopulmonary bypass.

In another embodiment, the methods of the present invention provideActivase®-Alteplase/tPA having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of acute myocardial infarction, acutemassive pulmonary embolism, or ischemic stroke.

In another embodiment, the methods of the present invention provideadenosine deaminase having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of severe combined immunodeficiencydisease.

In another embodiment, the methods of the present invention provideimmune globulin having additionally at least one CTP amino acid peptideon the N-terminus and one CTP amino acid peptide on the C-terminus forthe treatment of transplant recipients.

In another embodiment, the methods of the present invention provideimmune globulin is a CMV immune globulin. In another embodiment, themethods of the present invention provide glucocerebrosidase havingadditionally at least one CTP amino acid peptide on the N-terminus andone CTP amino acid peptide on the C-terminus for the treatment ofGaucher disease.

In another embodiment, the methods of the present invention provideLeukine-sargramostim/GM-CSF having additionally at least one CTP aminoacid peptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the stimulation of hematopoietic progenitor cells.

In another embodiment, the methods of the present invention provideG-CSF having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of Neutropenia. In another embodiment, the methods of thepresent invention provide Venoglobulin-S/IgG having additionally atleast one CTP amino acid peptide on the N-terminus and one CTP aminoacid peptide on the C-terminus for the treatment of immunodeficiencydiseases.

In another embodiment, the methods of the present invention provideProleukin-aldesleukin having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of renal carcinoma or metastatic melanoma.

In another embodiment, the methods of the present invention provideDNase having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of cystic fibrosis.

In another embodiment, the methods of the present invention provideFactor VIII having additionally at least one CTP amino acid peptide onthe N-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of Hemophilia A.

In another embodiment, the methods of the present invention providehelixate having additionally at least one CTP amino acid peptide on theN-terminus and one CTP amino acid peptide on the C-terminus for thetreatment of Hemophilia A.

In another embodiment, the methods of the present invention provideL-asparaginase having additionally at least one CTP amino acid peptideon the N-terminus and one CTP amino acid peptide on the C-terminus forthe treatment of acute lymphoblastic leukemia.

In another embodiment, the methods of the present invention provideWinRho SDF Rh IgG having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of Rh isoimmunization and immunethrombocytopenic purpura.

In another embodiment, the methods of the present invention provideRetavase retaplase/tPA having additionally at least one CTP amino acidpeptide on the N-terminus and one CTP amino acid peptide on theC-terminus for the treatment of acute myocardial infarction.

In another embodiment, the methods of the present invention provideFactor IX having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of Hemophilia B.

In another embodiment, the methods of the present invention provideFactor IX having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of Hemophilia B.

In another embodiment, the methods of the present invention provide FSHhaving additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forstimulation of ovulation during assisted reproduction.

In another embodiment, the methods of the present invention provideglobulin having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe prevention of respiratory syncytial virus disease.

In another embodiment, the methods of the present invention providefibrin having additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forwound management and hemostasis. In another embodiment, the methods ofthe present invention provide interleukin-11 having additionally atleast one CTP amino acid peptide on the N-terminus and at least one CTPamino acid peptide on the C-terminus for chemotherapy-inducedthrombocytopenia.

In another embodiment, the methods of the present invention providebecaplermin/PDGF having additionally at least one CTP amino acid peptideon the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of diabetic foot ulcers.

In another embodiment, the methods of the present invention providelepirudin/herudin having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for anticoagulation in heparin-induced thrombocytopenia.

In another embodiment, the methods of the present invention providesoluble TNF having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of rheumatoid arthritis.

In another embodiment, the methods of the present invention provideThymoglobulin having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of organ transplant rejection disease.

In another embodiment, the methods of the present invention provideFactor VIIa having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of hemophilia.

In another embodiment, the methods of the present invention provideinterferon alpha-2a having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of hairy cell leukemia and AIDS-relatedKaposi's sarcoma.

In another embodiment, the methods of the present invention provideinterferon alpha-2b having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of Hairy cell leukemia, genital warts,AIDS-related Kaposi's sarcoma, Hepatitis C, Hepatitis B, malignantmelanoma, and follicular lymphoma.

In another embodiment, the methods of the present invention provideinterferon alfa-N3 having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of genital warts.

In another embodiment, the methods of the present invention provideinterferon gamma-1b having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of chronic granulomatous disease.

In another embodiment, the methods of the present invention provideinterferon alfa n-1 having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of Hepatitis C infection.

In another embodiment, the methods of the present invention provideInterleukin-2 having additionally at least one CTP amino acid peptide onthe N-terminus and at least one CTP amino acid peptide on the C-terminusfor the treatment of renal carcinoma and metastatic melanoma.

In another embodiment, the methods of the present invention provideinterferon beta-1b having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for the treatment of multiple sclerosis.

In another embodiment, the methods of the present invention provide hGHhaving additionally at least one CTP amino acid peptide on theN-terminus and at least one CTP amino acid peptide on the C-terminus forthe treatment of wasting disease, AIDS, cachexia, or hGH deficiency.

In another embodiment, the methods of the present invention provide anOKT3 monoclonal antibody having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for organ transplant.

In another embodiment, the methods of the present invention provide aReo monoclonal antibody having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for prevention of complications from coronary interventionand angioplasty.

In another embodiment, the methods of the present invention provide amonoclonal antibody having additionally at least one CTP amino acidpeptide on the N-terminus and at least one CTP amino acid peptide on theC-terminus for treating colorectal cancer, Non-Hodgkin's lymphoma,kidney transplant rejection, metastatic breast cancer, or the preventionof respiratory syncytial virus disease.

In some embodiments, the CTP sequence modification is advantageous inpermitting lower dosages to be used.

In some embodiments, “polypeptide” or “protein” as used hereinencompasses native polypeptides (either degradation products,synthetically synthesized polypeptides or recombinant polypeptides) andpeptidomimetics (typically, synthetic polypeptides), as well as peptoidsand semipeptoids which are polypeptide analogs, which have, in someembodiments, modifications rendering the polypeptides even more stablewhile in a body or more capable of penetrating into cells.

In some embodiments, modifications include, but are not limited to Nterminus modification, C terminus modification, polypeptide bondmodification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O,O═C—NH, CH2-O, CH2-CH₂, S═C—NH, CH═CH or CF═CH, backbone modifications,and residue modification. Methods for preparing peptidomimetic compoundsare well known in the art and are specified, for example, inQuantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. ChoplinPergamon Press (1992), which is incorporated by reference as if fullyset forth herein. Further details in this respect are providedhereinbelow.

In some embodiments, polypeptide bonds (—CO—NH—) within the polypeptideare substituted. In some embodiments, the polypeptide bonds aresubstituted by N-methylated bonds (—N(CH3)-CO—). In some embodiments,the polypeptide bonds are substituted by ester bonds(—C(R)H—C—O—O—C(R)—N—). In some embodiments, the polypeptide bonds aresubstituted by ketomethylen bonds (—CO—CH2-). In some embodiments, thepolypeptide bonds are substituted by α-aza bonds (—NH—N(R)—CO—), whereinR is any alkyl, e.g., methyl, carba bonds (—CH2-NH—). In someembodiments, the polypeptide bonds are substituted by hydroxyethylenebonds (—CH(OH)—CH2-). In some embodiments, the polypeptide bonds aresubstituted by thioamide bonds (—CS—NH—). In some embodiments, thepolypeptide bonds are substituted by olefinic double bonds (—CH═CH—). Insome embodiments, the polypeptide bonds are substituted by retro amidebonds (—NH—CO—). In some embodiments, the polypeptide bonds aresubstituted by polypeptide derivatives (—N(R)—CH2-CO—), wherein R is the“normal” side chain, naturally presented on the carbon atom. In someembodiments, these modifications occur at any of the bonds along thepolypeptide chain and even at several (2-3 bonds) at the same time.

In some embodiments, natural aromatic amino acids of the polypeptidesuch as Trp, Tyr and Phe, are substituted for synthetic non-natural acidsuch as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylatedderivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. Insome embodiments, the polypeptides of the present invention include oneor more modified amino acids or one or more non-amino acid monomers(e.g. fatty acid, complex carbohydrates etc).

In one embodiment, “amino acid” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. In one embodiment,“amino acid” includes both D- and L-amino acids.

In some embodiments, the polypeptides of the present invention areutilized in therapeutics which requires the polypeptides to be in asoluble form. In some embodiments, the polypeptides of the presentinvention include one or more non-natural or natural polar amino acids,including, but not limited to, serine and threonine, which are capableof increasing polypeptide solubility due to their hydroxyl-containingside chain.

In some embodiments, the cytokines of the present invention are utilizedin a linear form, although it will be appreciated by one skilled in theart that in cases where cyclicization does not severely interfere withcytokines characteristics, cyclic forms of the cytokines can also beutilized.

In some embodiments, the cytokines of present invention arebiochemically synthesized such as by using standard solid phasetechniques. In some embodiments, these biochemical methods includeexclusive solid phase synthesis, partial solid phase synthesis, fragmentcondensation, or classical solution synthesis. In some embodiments,these methods are used when the cytokines are relatively short (about5-15kDa) and/or when it cannot be produced by recombinant techniques(i.e., not encoded by a nucleic acid sequence) and therefore involvesdifferent chemistry.

In some embodiments, solid phase cytokines synthesis procedures are wellknown to one skilled in the art and further described by John MorrowStewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2″Ed., Pierce Chemical Company, 1984). In some embodiments, syntheticpolypeptides are purified by preparative high performance liquidchromatography [Creighton T. (1983) Proteins, structures and molecularprinciples. WH Freeman and Co. N.Y.], the composition of which can beconfirmed via amino acid sequencing by methods known to one skilled inthe art.

In some embodiments, recombinant protein techniques are used to generatethe cytokines of the present invention. In some embodiments, recombinantprotein techniques are used for generation of relatively longpolypeptides (e.g., longer than 18-25 amino acid). In some embodiments,recombinant protein techniques are used for the generation of largeamounts of the cytokines of the present invention. In some embodiments,recombinant techniques are described by Bitter et al., (1987) Methods inEnzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol.185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al.(1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 andBrogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol.Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for PlantMolecular Biology, Academic Press, NY, Section V111, pp 421-463.

In another embodiment, cytokines of the present invention aresynthesized using a polynucleotide encoding a polypeptide of the presentinvention. In some embodiments, the polynucleotide encoding cytokines ofthe present invention is ligated into an expression vector, comprising atranscriptional control of a cis-regulatory sequence (e.g., promotersequence). In some embodiments, the cis-regulatory sequence is suitablefor directing constitutive expression of the cytokines of the presentinvention. In some embodiments, the cis-regulatory sequence is suitablefor directing tissue-specific expression of the cytokines of the presentinvention. In some embodiments, the cis-regulatory sequence is suitablefor directing inducible expression of the cytokines of the presentinvention.

In some embodiments, tissue-specific promoters suitable for use with thepresent invention include sequences which are functional in a specificcell population. Examples include, but are not limited to, promoterssuch as albumin that is liver specific [Pinkert et al., (1987) GenesDev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv.Immunol. 43:235-275]; in particular promoters of T-cell receptors[Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerjiet al. (1983) Cell 33729-740], neuron-specific promoters such as theneurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985)Science 230:912-916] or mammary gland-specific promoters such as themilk whey promoter (U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Inducible promoters suitable for use with thepresent invention include, for example, the tetracycline-induciblepromoter (Srour, M. A., et al., 2003. Thromb. Haemost. 90: 398-405).

In one embodiment, the phrase “a polynucleotide” refers to a single ordouble stranded nucleic acid sequence which be isolated and provided inthe form of an RNA sequence, a complementary polynucleotide sequence(cDNA), a genomic polynucleotide sequence and/or a compositepolynucleotide sequences (e.g., a combination of the above).

In one embodiment, “complementary polynucleotide sequence” refers to asequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.In one embodiment, the sequence can be subsequently amplified in vivo orin vitro using a DNA polymerase.

In one embodiment, “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

In one embodiment, “composite polynucleotide sequence” refers to asequence, which is at least partially complementary and at leastpartially genomic. In one embodiment, a composite sequence can includesome exonal sequences required to encode the polypeptide of the presentinvention, as well as some intronic sequences interposing therebetween.In one embodiment, the intronic sequences can be of any source,including of other genes, and typically will include conserved splicingsignal sequences. In one embodiment, intronic sequences include cisacting expression regulatory elements.

In one embodiment, the polynucleotides of the present invention furthercomprise a signal sequence encoding a signal peptide for the secretionof the cytokines of the present invention. In some embodiments, signalsequences include, but are not limited to the endogenous signal sequencefor EPO as set forth in SEQ ID NO: 19 or the endogenous signal sequencefor IFN-β1 as set forth in SEQ ID NO: 64. In another embodiment, thesignal sequence is N-terminal to the CTP sequence that is in turnN-terminal to the polypeptide sequence of interest; e.g. the sequence is(a) signal sequence-(b) CTP-(c) sequence-of-interest-(d) optionally, 1or more additional CTP sequences. In another embodiment, 1 or more CTPsequences is inserted between the signal sequence of a polypeptidesequence of interest and the polypeptide sequence of interest itself,thus interrupting the wild-type sequence of interest. Each possibilityrepresents a separate embodiment of the present invention.

In one embodiment, following expression and secretion, the signalpeptides are cleaved from the precursor proteins resulting in the matureproteins.

In some embodiments, polynucleotides of the present invention areprepared using PCR techniques as described in Example 1, or any othermethod or procedure known to one skilled in the art. In someembodiments, the procedure involves the ligation of two different DNAsequences (See, for example, “Current Protocols in Molecular Biology”,eds. Ausubel et al., John Wiley & Sons, 1992).

In one embodiment, polynucleotides of the present invention are insertedinto expression vectors (i.e., a nucleic acid construct) to enableexpression of the recombinant polypeptide. In one embodiment, theexpression vector of the present invention includes additional sequenceswhich render this vector suitable for replication and integration inprokaryotes. In one embodiment, the expression vector of the presentinvention includes additional sequences which render this vectorsuitable for replication and integration in eukaryotes. In oneembodiment, the expression vector of the present invention includes ashuttle vector which renders this vector suitable for replication andintegration in both prokaryotes and eukaryotes. In some embodiments,cloning vectors comprise transcription and translation initiationsequences (e.g., promoters, enhancers) and transcription and translationterminators (e.g., polyadenylation signals).

In one embodiment, a variety of prokaryotic or eukaryotic cells can beused as host-expression systems to express the cytokines of the presentinvention. In some embodiments, these include, but are not limited to,microorganisms, such as bacteria transformed with a recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorcontaining the polypeptide coding sequence; yeast transformed withrecombinant yeast expression vectors containing the polypeptide codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors, such asTi plasmid, containing the polypeptide coding sequence.

In some embodiments, non-bacterial expression systems are used (e.g.mammalian expression systems such as CHO cells) to express the cytokinesof the present invention. In one embodiment, the expression vector usedto express polynucleotides of the present invention in mammalian cellsis pCI-DHFR vector comprising a CMV promoter and a neomycin resistancegene. Construction of the pCI-dhfr vector is described, according to oneembodiment, in Example 1.

In some embodiments, in bacterial systems of the present invention, anumber of expression vectors can be advantageously selected dependingupon the use intended for the polypeptide expressed. In one embodiment,large quantities of polypeptide are desired. In one embodiment, vectorsthat direct the expression of high levels of the protein product,possibly as a fusion with a hydrophobic signal sequence, which directsthe expressed product into the periplasm of the bacteria or the culturemedium where the protein product is readily purified are desired. In oneembodiment, certain fusion proteins are engineered with a specificcleavage site to aid in recovery of the polypeptide. In one embodiment,vectors adaptable to such manipulation include, but are not limited to,the pET series of E. coli expression vectors [Studier et al., Methods inEnzymol. 185:60-89 (1990)].

In one embodiment, yeast expression systems are used. In one embodiment,a number of vectors containing constitutive or inducible promoters canbe used in yeast as disclosed in U.S. Pat. No. 5,932,447. In anotherembodiment, vectors which promote integration of foreign DNA sequencesinto the yeast chromosome are used.

In one embodiment, the expression vector of the present invention canfurther include additional polynucleotide sequences that allow, forexample, the translation of several proteins from a single mRNA such asan internal ribosome entry site (IRES) and sequences for genomicintegration of the promoter-chimeric polypeptide.

In some embodiments, mammalian expression vectors include, but are notlimited to, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2,pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB,pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

In some embodiments, expression vectors containing regulatory elementsfrom eukaryotic viruses such as retroviruses are used by the presentinvention. SV40 vectors include pSVT7 and pMT2. In some embodiments,vectors derived from bovine papilloma virus include pBV-1MTHA, andvectors derived from Epstein Barr virus include pHEBO, and p205. Otherexemplary vectors include pMSG, pAV009/A⁺, pMT010/A⁺, pMAMneo-5,baculovirus pDSVE, and any other vector allowing expression of proteinsunder the direction of the SV-40 early promoter, SV-40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

In some embodiments, recombinant viral vectors are useful for in vivoexpression of the cytokines of the present invention, since they offeradvantages such as lateral infection and targeting specificity. In oneembodiment, lateral infection is inherent in the life cycle of, forexample, retrovirus and is the process by which a single infected cellproduces many progeny virions that bud off and infect neighboring cells.In one embodiment, the result is that a large area becomes rapidlyinfected, most of which was not initially infected by the original viralparticles. In one embodiment, viral vectors are produced that are unableto spread laterally. In one embodiment, this characteristic can beuseful if the desired purpose is to introduce a specified gene into onlya localized number of targeted cells.

In one embodiment, various methods can be used to introduce theexpression vector of the present invention into cells. Such methods aregenerally described in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Springs Harbor Laboratory, New York (1989, 1992), inAusubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRCPress, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press,Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectorsand Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at.[Biotechniques 4 (6): 504-512, 1986] and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

In some embodiments, introduction of nucleic acid by viral infectionoffers several advantages over other methods such as lipofection andelectroporation, since higher transfection efficiency can be obtaineddue to the infectious nature of viruses.

In one embodiment, it will be appreciated that the cytokines of thepresent invention can also be expressed from a nucleic acid constructadministered to the individual employing any suitable mode ofadministration, described hereinabove (i.e., in vivo gene therapy). Inone embodiment, the nucleic acid construct is introduced into a suitablecell via an appropriate gene delivery vehicle/method (transfection,transduction, homologous recombination, etc.) and an expression systemas needed and then the modified cells are expanded in culture andreturned to the individual (i.e., ex vivo gene therapy).

In one embodiment, in vivo gene therapy using a cytokine has beenattempted in animal models such as rodents [Bohl et al., Blood. 2000;95:2793-2798], primates [Gao et al., Blood, 2004, Volume 103, Number 9]and has proven successful in human clinical trials for patients withchronic renal failure [Lippin et al Blood 2005, 106, Number 7].

In one embodiment, plant expression vectors are used. In one embodiment,the expression of a polypeptide coding sequence is driven by a number ofpromoters. In some embodiments, viral promoters such as the ³⁵S RNA and19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)],or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311(1987)] are used. In another embodiment, plant promoters are used suchas, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J.3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] orheat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley etal., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructsare introduced into plant cells using Ti plasmid, Ri plasmid, plantviral vectors, direct DNA transformation, microinjection,electroporation and other techniques well known to the skilled artisan.See, for example, Weissbach & Weissbach [Methods for Plant MolecularBiology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Otherexpression systems such as insects and mammalian host cell systems,which are well known in the art, can also be used by the presentinvention.

It will be appreciated that other than containing the necessary elementsfor the transcription and translation of the inserted coding sequence(encoding the polypeptide), the expression construct of the presentinvention can also include sequences engineered to optimize stability,production, purification, yield or activity of the expressedpolypeptide.

Various methods, in some embodiments, can be used to introduce theexpression vector of the present invention into the host cell system. Insome embodiments, such methods are generally described in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Springs HarborLaboratory, New York (1989, 1992), in Ausubel et al., Current Protocolsin Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Changet al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vegaet al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Butterworths, BostonMass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] andinclude, for example, stable or transient transfection, lipofection,electroporation and infection with recombinant viral vectors. Inaddition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 forpositive-negative selection methods.

In some embodiments, transformed cells are cultured under effectiveconditions, which allow for the expression of high amounts ofrecombinant polypeptide. In some embodiments, effective cultureconditions include, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Inone embodiment, an effective medium refers to any medium in which a cellis cultured to produce the recombinant polypeptide of the presentinvention. In some embodiments, a medium typically includes an aqueoussolution having assimilable carbon, nitrogen and phosphate sources, andappropriate salts, minerals, metals and other nutrients, such asvitamins. In some embodiments, cells of the present invention can becultured in conventional fermentation bioreactors, shake flasks, testtubes, microtiter dishes and petri plates. In some embodiments,culturing is carried out at a temperature, pH and oxygen contentappropriate for a recombinant cell. In some embodiments, culturingconditions are within the expertise of one of ordinary skill in the art.

In some embodiments, depending on the vector and host system used forproduction, resultant cytokines of the present invention either remainwithin the recombinant cell, secreted into the fermentation medium,secreted into a space between two cellular membranes, such as theperiplasmic space in E. coli; or retained on the outer surface of a cellor viral membrane.

In one embodiment, following a predetermined time in culture, recoveryof the recombinant polypeptide is effected.

In one embodiment, the phrase “recovering the recombinant polypeptide”used herein refers to collecting the whole fermentation mediumcontaining the polypeptide and need not imply additional steps ofseparation or purification.

In one embodiment, cytokines of the present invention are purified usinga variety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.

In one embodiment, to facilitate recovery, the expressed coding sequencecan be engineered to encode the polypeptide of the present invention andfused cleavable moiety. In one embodiment, a fusion protein can bedesigned so that the polypeptide can be readily isolated by affinitychromatography; e.g., by immobilization on a column specific for thecleavable moiety. In one embodiment, a cleavage site is engineeredbetween the polypeptide and the cleavable moiety, and the polypeptidecan be released from the chromatographic column by treatment with anappropriate enzyme or agent that specifically cleaves the fusion proteinat this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988);and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

In one embodiment, the polypeptide of the present invention is retrievedin “substantially pure” form.

In one embodiment, the phrase “substantially pure” refers to a puritythat allows for the effective use of the protein in the applicationsdescribed herein.

In one embodiment, the polypeptide of the present invention can also besynthesized using in vitro expression systems. In one embodiment, invitro synthesis methods are well known in the art and the components ofthe system are commercially available.

In one embodiment, production of CTP-cytokine polypeptides usingrecombinant DNA technology is illustrated in Example 1.

In some embodiments, the recombinant polypeptides are synthesized andpurified; their therapeutic efficacy can be assayed either in vivo or invitro. In one embodiment, the binding activities of the recombinantcytokines of the present invention can be ascertained using variousassays as described in Examples 2-6 and 8-9.

In another embodiment, in vitro binding activity is ascertained bymeasuring the ability of the cytokine as described herein as well aspharmaceutical compositions comprising the same to treat diseases suchas cancers such as hairy cell leukemia, malignant melanoma, Kaposi'ssarcoma, bladder cancer, chronic myelocytic leukemia, kidney cancer,carcinoid tumors, non-Hodgkin's lymphoma, ovarian cancer, and skincancers (for interferons). In one embodiment, in vivo activity isdeduced by analyzing haematocrit levels (FIGS. 3-5) and/or as apercentage of reticulocytes (for EPO). In another embodiment, in vivoactivity is deduced by known measures of the disease that is beingtreated.

In some embodiments, the phrase “erythropoietin-associated conditions”refers to any condition associated with below normal, abnormal, orinappropriate modulation of erythropoietin. In some embodiments, levelsof erythropoietin associated with such conditions are determined by anymeasure accepted and utilized by those of skill in the art. In someembodiments, erythropoietin-associated conditions typically includeanemic conditions.

In some embodiments, “anemic conditions” refers to any condition,disease, or disorder associated with anemia. In some embodiments, anemicconditions include, but are not limited to, aplastic anemia, autoimmunehemolytic anemia, bone marrow transplantation, Churg-Strauss syndrome,Diamond Blackfan anemia, Fanconi's anemia, Felty syndrome, graft versushost disease, hematopoietic stem cell transplantation, hemolytic uremicsyndrome, myelodysplasic syndrome, nocturnal paroxysmal hemoglobinuria,osteomyelofibrosis, pancytopenia, pure red-cell aplasia, purpuraSchoenlein-Henoch, sideroblastic anemia, refractory anemia with excessof blasts, rheumatoid arthritis, Shwachman syndrome, sickle celldisease, thalassemia major, thalassemia minor, thrombocytopenic purpura,etc.

In one embodiment, the present invention comprises CTP-hGH-CTP-CTPpolypeptides. In one embodiment, recombinant DNA technology methods areused for the production of CTP-hGH-CTP-CTP polypeptides as illustratedin Example 7. In one embodiment, the therapeutic efficacy of theCTP-hGH-CTP-CTP polypeptides of the present invention is assayed invivo. In one embodiment, the therapeutic efficacy of the CTP-hGH-CTP-CTPpolypeptides of the present invention is assayed in vitro. In oneembodiment, the binding activities of the recombinant hGH polypeptidesof the present invention are measured using Nb2 (a prolactin-dependentrat lymphoma cell line (ECACC Cell Bank)) or a FCD-Pl murine cell line,previously transfected with human growth hormone receptor. In oneembodiment, binding of hGH to these receptors induces cellproliferation, which, in one embodiment, is measured by the levels ofMTT cellular stain as a function of hGH activity. In one embodiment, invivo activity is deduced by measuring weight gain over time in treatedgrowth hormone-deficient animals.

In some embodiments, human growth hormone polypeptides of the presentinvention can be used to treat a subject, with conditions related togrowth and weight, such as a growth deficiency disorder, AIDS wasting,aging, impaired immune function of HIV-infected subjects, a catabolicillness, surgical recovery, a congestive cardiomyopathy, livertransplantation, liver regeneration after hepatectomy, chronic renalfailure, renal osteodystrophy, osteoporosis,achondroplasia/hypochondroplasia, skeletal dysplasia, a chronicinflammatory or nutritional disorder such as Crohn's disease, shortbowel syndrome, juvenile chronic arthritis, cystic fibrosis, maleinfertility, X-linked hypophosphatemic rickets, Down's syndrome, Spinabifida, Noonan Syndrome, obesity, impaired muscle strength andfibromyalgia.

In some embodiments, interferon polypeptides of the present inventionare used to treat a subject, with a variety of conditions such as hairycell leukemia (HCL), Kaposi's sarcoma (KS), chronic myelogenous leukemia(CML), chronic Hepatitis C(CHC), condylomata accuminata (CA), chronicHepatitis B, malignant melanoma, follicular non-Hodgkin's lymphoma,multiple sclerosis, chronic granulomatous disease, Mycobacterium aviumcomplex (MAC), pulmonary fibrosis, osteoarthritis, and osteoporosis.

In another embodiment, polypeptides of the present invention comprisingIFN α-2a as well as pharmaceutical compositions comprising the same areindicated for hairy cell leukemia (HCL), acquired immune deficiencysyndrome (AIDS)-related Kaposi's sarcoma (KS), chronic-phasePhiladelphia (Ph) chromosome-positive chronic myelogenous leukemia (CML)and chronic Hepatitis C(CHC). IFN α-2a dosage varies depending on theindication. In another embodiment, the effectiveness of IFN α-2a as anantineoplastic, immunomodulator to and antiviral agent has beenestablished.

In another embodiment, polypeptides of the present invention comprisingIFN α1b as well as pharmaceutical compositions comprising the same areindicated for HCL, AIDS-related Kaposi's sarcoma and CHC. It is alsoindicated for condylomata accuminata (CA), chronic Hepatitis B,malignant melanoma and follicular non-Hodgkin's lymphoma. IFN α-2bdosage varies depending on its indication of usage.

In another embodiment, polypeptides of the present invention comprisinga cytokine are administered in a dose of 1-90 micrograms in 0.1-5 mlsolution. In another embodiment, polypeptides of the present inventioncomprising a cytokine are administered in a dose of 1-50 micrograms in0.1-5 ml solution. In another embodiment, polypeptides of the presentinvention comprising a cytokine are administered in a dose of 1-25micrograms in 0.1-5 ml solution. In another embodiment, polypeptides ofthe present invention comprising a cytokine are administered in a doseof 50-90 micrograms in 0.1-5 ml solution. In another embodiment,polypeptides of the present invention comprising a cytokine areadministered in a dose of 10-50 micrograms in 0.1-5 ml solution.

In another embodiment, polypeptides of the present invention comprisinga cytokine are administered in a dose of 1-90 micrograms in 0.1-5 mlsolution by intramuscular (i.m.) injection, subcutaneous (s.c.)injection, or intravenous (i.v.) injection once a week. In anotherembodiment, polypeptides of the present invention comprising a cytokineare administered in a dose of 1-90 micrograms in 0.1-5 ml solution byintramuscular (i.m.) injection, subcutaneous (s.c.) injection, orintravenous (i.v.) injection twice a week. In another embodiment,polypeptides of the present invention comprising a cytokine areadministered in a dose of 1-90 micrograms in 0.1-5 ml solution byintramuscular (i.m.) injection, subcutaneous (s.c.) injection, orintravenous (i.v.) injection three times a week. In another embodiment,polypeptides of the present invention comprising a cytokine areadministered in a dose of 1-90 micrograms in 0.1-5 ml solution byintramuscular (i.m.) injection, subcutaneous (s.c.) injection, orintravenous (i.v.) injection once every two weeks. In anotherembodiment, polypeptides of the present invention comprising a cytokineare administered in a dose of 1-90 micrograms in 0.1-5 ml solution byintramuscular (i.m.) injection, subcutaneous (s.c.) injection, orintravenous (i.v.) injection once every 17 days. In another embodiment,polypeptides of the present invention comprising a cytokine areadministered in a dose of 1-90 micrograms in 0.1-5 ml solution byintramuscular (i.m.) injection, subcutaneous (s.c.) injection, orintravenous (i.v.) injection once every 19 days.

In another embodiment, polypeptides of the present invention compriserecombinant cytokines. In another embodiment, polypeptides of thepresent invention comprise a recombinant IFN-β. In another embodiment,polypeptides of the present invention comprise a recombinant IFN-α. Inanother embodiment, various recombinant IFN are known to one of skill inthe art.

In another embodiment, protein drugs of molecular weight lower than50,000 daltons, such as interferons, are in general short-lived speciesin vivo, having short circulatory half-lives of several hours. Inanother embodiment, the subcutaneous route of administration in generalprovides slower release into the circulation. In another embodiment, theCTP-modified polypeptide of the invention prolongs the half-life ofprotein drugs of molecular weight lower than 50,000 daltons, such asinterferons. In another embodiment, the CTP-modified polypeptide of theinvention enables interferons to exert their beneficial effects for alonger period of time.

In another embodiment, the immunogenicity of a CTP-modified polypeptidecomprising a cytokine is equal to an isolated cytokine. In anotherembodiment, the immunogenicity of a CTP-modified polypeptide comprisinga cytokine is comparable to an isolated cytokine. In another embodiment,modifying a cytokine as described herein with CTP peptides reduces theimmunogenicity of the cytokine. In another embodiment, the CTP-modifiedpolypeptide comprising a cytokine is as active as an isolated cytokineprotein. In another embodiment, the CTP-modified polypeptide comprisinga cytokine is more active than an isolated cytokine. In anotherembodiment, the CTP-modified polypeptide comprising a cytokine maximizesthe cytokine's protective ability against degradation while minimizingreductions in bioactivity.

In another embodiment, the cytokine of the present invention can beprovided to the individual per se. In one embodiment, the cytokine ofthe present invention can be provided to the individual as part of apharmaceutical composition where it is mixed with a pharmaceuticallyacceptable carrier.

In another embodiment, a “pharmaceutical composition” refers to apreparation of one or more of the active ingredients described hereinwith other chemical components such as physiologically suitable carriersand excipients. The purpose of a pharmaceutical composition is tofacilitate administration of a compound to an organism.

In another embodiment, “active ingredient” refers to the polypeptidesequence of interest, which is accountable for the biological effect.

In another embodiment, any of the compositions of this invention willcomprise at least two CTP sequences bound to a cytokine of interest, inany form. In one embodiment, the present invention provides combinedpreparations. In one embodiment, “a combined preparation” definesespecially a “kit of parts” in the sense that the combination partnersas defined above can be dosed independently or by use of different fixedcombinations with distinguished amounts of the combination partnersi.e., simultaneously, concurrently, separately or sequentially. In someembodiments, the parts of the kit of parts can then, e.g., beadministered simultaneously or chronologically staggered, that is, atdifferent time points and with equal or different time intervals for anypart of the kit of parts. The ratio of the total amounts of thecombination partners, in some embodiments, can be administered in thecombined preparation. In one embodiment, the combined preparation can bevaried, e.g., in order to cope with the needs of a patient subpopulationto be treated or the needs of the single patient which different needscan be due to a particular disease, severity of a disease, age, sex, orbody weight as can be readily made by a person skilled in the art.

In another embodiment, the phrases “physiologically acceptable carrier”and “pharmaceutically acceptable carrier” which can be usedinterchangeably refer to a carrier or a diluent that does not causesignificant irritation to an organism and does not abrogate thebiological activity and properties of the administered compound. Anadjuvant is included under these phrases. In one embodiment, one of theingredients included in the pharmaceutically acceptable carrier can befor example polyethylene glycol (PEG), a biocompatible polymer with awide range of solubility in both organic and aqueous media (Mutter etal. (1979).

In another embodiment, “excipient” refers to an inert substance added toa pharmaceutical composition to further facilitate administration of anactive ingredient. In one embodiment, excipients include calciumcarbonate, calcium phosphate, various sugars and type of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs are found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

In another embodiment, suitable routes of administration, for example,include oral, rectal, transmucosal, transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

In another embodiment, the preparation is administered in a local ratherthan systemic manner, for example, via injection of the preparationdirectly into a specific region of a patient's body.

Various embodiments of dosage ranges are contemplated by this invention.The dosage of the cytokine of the present invention, in one embodiment,is in the range of 0.005-100 mg/day. In another embodiment, the dosageis in the range of 0.005-5 mg/day. In another embodiment, the dosage isin the range of 0.01-50 mg/day. In another embodiment, the dosage is inthe range of 0.1-20 mg/day. In another embodiment, the dosage is in therange of 0.1-10 mg/day. In another embodiment, the dosage is in therange of 0.01-5 mg/day. In another embodiment, the dosage is in therange of 0.001-0.01 mg/day. In another embodiment, the dosage is in therange of 0.001-0.1 mg/day. In another embodiment, the dosage is in therange of 0.1-5 mg/day. In another embodiment, the dosage is in the rangeof 0.5-50 mg/day. In another embodiment, the dosage is in the range of0.2-15 mg/day. In another embodiment, the dosage is in the range of0.8-65 mg/day. In another embodiment, the dosage is in the range of 1-50mg/day. In another embodiment, the dosage is in the range of 5-10mg/day. In another embodiment, the dosage is in the range of 8-15mg/day. In another embodiment, the dosage is in a range of 10-20 mg/day.In another embodiment, the dosage is in the range of 20-40 mg/day. Inanother embodiment, the dosage is in a range of 60-120 mg/day. Inanother embodiment, the dosage is in the range of 12-40 mg/day. Inanother embodiment, the dosage is in the range of 40-60 mg/day. Inanother embodiment, the dosage is in a range of 50-100 mg/day. Inanother embodiment, the dosage is in a range of 1-60 mg/day. In anotherembodiment, the dosage is in the range of 15-25 mg/day. In anotherembodiment, the dosage is in the range of 5-10 mg/day. In anotherembodiment, the dosage is in the range of 55-65 mg/day.

In another embodiment, a polypeptide comprising a cytokine and CTP unitsis formulated in an intranasal dosage form. In another embodiment, apolypeptide comprising a cytokine and CTP units is formulated in aninjectable dosage form. In another embodiment, a polypeptide comprisinga cytokine and CTP units is administered to a subject in a dose rangingfrom 0.0001 mg to 0.6 mg. In another embodiment, a polypeptidecomprising a cytokine and CTP units is administered to a subject in adose ranging from 0.001 mg to 0.005 mg. In another embodiment, apolypeptide comprising a cytokine and CTP units is administered to asubject in a dose ranging from 0.005 mg to 0.01 mg. In anotherembodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject in a dose ranging from 0.01 mg to 0.3 mg. Inanother embodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject in a dose in a dose ranging from 0.2 mg to 0.6mg.

In another embodiment, a polypeptide comprising a cytokine and CTP unitsis administered to a subject in a dose ranging from 1-100 micrograms. Inanother embodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject in a dose ranging from 10-80 micrograms. Inanother embodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject in a dose ranging from 20-60 micrograms. Inanother embodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject in a dose ranging from 10-50 micrograms. Inanother embodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject in a dose ranging from 40-80 micrograms. Inanother embodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject in a dose ranging from 10-30 micrograms. Inanother embodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject in a dose ranging from 30-60 micrograms.

In another embodiment, a polypeptide comprising a cytokine and CTP unitsis administered to a subject in a dose ranging from 0.2 mg to 2 mg. Inanother embodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject in a dose ranging from 2 mg to 6 mg. Inanother embodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject in a dose ranging from 4 mg to 10 mg. Inanother embodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject in a dose ranging from 5 mg to 15 mg.

In another embodiment, a polypeptide comprising a cytokine and CTP unitsis injected into the muscle (intramuscular injection). In anotherembodiment, a polypeptide comprising a cytokine and CTP units isinjected below the skin (subcutaneous injection). In another embodiment,a polypeptide comprising an IFN protein and CTP units is injected intothe muscle. In another embodiment, a polypeptide comprising an IFNprotein and CTP units is injected below the skin.

In another embodiment, the methods of the invention include increasingthe compliance in the use of cytokine therapy, comprising providing to asubject in need thereof, a polypeptide comprising a cytokine, onechorionic gonadotrophin carboxy terminal peptide (CTP) attached to anamino terminus of the cytokine, and two chorionic gonadotrophin carboxyterminal peptides attached to a carboxy terminus of the cytokine,thereby increasing compliance in the use of cytokine therapy.

In another embodiment, the methods of the invention include increasingthe compliance of patients afflicted with chronic illnesses that are inneed of a cytokine therapy. In another embodiment, the methods of theinvention enable reduction in the dosing frequency of a cytokine bymodifying the cytokine with CTPs as described hereinabove. In anotherembodiment, the term compliance comprises adherence. In anotherembodiment, the methods of the invention include increasing thecompliance of patients in need of a cytokine therapy by reducing thefrequency of administration of the cytokine. In another embodiment,reduction in the frequency of administration of the cytokine is achieveddue to the CTP modifications which render the CTP-modified cytokine morestable. In another embodiment, reduction in the frequency ofadministration of the cytokine is achieved as a result of increasingT_(1/2) of the cytokine. In another embodiment, reduction in thefrequency of administration of the cytokine is achieved as a result ofincreasing the clearance time of the cytokine. In another embodiment,reduction in the frequency of administration of the cytokine is achievedas a result of increasing the AUC measure of the cytokine.

In another embodiment, a polypeptide comprising a cytokine and CTP unitsis administered to a subject once a day. In another embodiment, apolypeptide comprising a cytokine and CTP units is administered to asubject once every two days. In another embodiment, a polypeptidecomprising a cytokine and CTP units is administered to a subject onceevery three days. In another embodiment, a polypeptide comprising acytokine and CTP units is administered to a subject once every fourdays. In another embodiment, a polypeptide comprising a cytokine and CTPunits is administered to a subject once every five days. In anotherembodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject once every six days. In another embodiment, apolypeptide comprising a cytokine and CTP units is administered to asubject once every week. In another embodiment, a polypeptide comprisinga cytokine and CTP units is administered to a subject once every 7-14days. In another embodiment, a polypeptide comprising a cytokine and CTPunits is administered to a subject once every 10-20 days. In anotherembodiment, a polypeptide comprising a cytokine and CTP units isadministered to a subject once every 5-15 days. In another embodiment, apolypeptide comprising an a cytokine and CTP units is administered to asubject once every 15-30 days.

In another embodiment, the dosage is in a range of 50-500 mg/day. Inanother embodiment, the dosage is in a range of 50-150 mg/day. Inanother embodiment, the dosage is in a range of 100-200 mg/day. Inanother embodiment, the dosage is in a range of 150-250 mg/day. Inanother embodiment, the dosage is in a range of 200-300 mg/day. Inanother embodiment, the dosage is in a range of 250-400 mg/day. Inanother embodiment, the dosage is in a range of 300-500 mg/day. Inanother embodiment, the dosage is in a range of 350-500 mg/day.

In one embodiment, the dosage is 20 mg/day. In one embodiment, thedosage is 30 mg/day. In one embodiment, the dosage is 40 mg/day. In oneembodiment, the dosage is 50 mg/day. In one embodiment, the dosage is0.01 mg/day. In another embodiment, the dosage is 0.1 mg/day. In anotherembodiment, the dosage is 1 mg/day. In another embodiment, the dosage is0.530 mg/day. In another embodiment, the dosage is 0.05 mg/day. Inanother embodiment, the dosage is 10 mg/day. In another embodiment, thedosage is 20-70 mg/day. In another embodiment, the dosage is 5 mg/day.

In another embodiment, the dosage is 1-90 mg/day. In another embodiment,the dosage is 1-90 mg/2 days. In another embodiment, the dosage is 1-90mg/3 days. In another embodiment, the dosage is 1-90 mg/4 days. Inanother embodiment, the dosage is 1-90 mg/5 days. In another embodiment,the dosage is 1-90 mg/6 days. In another embodiment, the dosage is 1-90mg/week. In another embodiment, the dosage is 1-90 mg/9 days. In anotherembodiment, the dosage is 1-90 mg/11 days. In another embodiment, thedosage is 1-90 mg/14 days.

In another embodiment, the cytokine dosage is 10-50 mg/day. In anotherembodiment, the dosage is 10-50 mg/2 days. In another embodiment, thedosage is 10-50 mg/3 days. In another embodiment, the dosage is 10-50mg/4 days. In another embodiment, the dosage is 10-50 micrograms mg/5days. In another embodiment, the dosage is 10-50 mg/6 days. In anotherembodiment, the dosage is 10-50 mg/week. In another embodiment, thedosage is 10-50 mg/9 days. In another embodiment, the dosage is 10-50mg/11 days. In another embodiment, the dosage is 10-50 mg/14 days.

Oral administration, in one embodiment, comprises a unit dosage formcomprising tablets, capsules, lozenges, chewable tablets, suspensions,emulsions and the like. Such unit dosage forms comprise a safe andeffective amount of the desired cytokine of the invention, each of whichis, in one embodiment, from about 0.7 or 3.5 mg to about 280 mg/70 kg,or, in another embodiment, about 0.5 or 10 mg to about 210 mg/70 kg. Thepharmaceutically acceptable carriers suitable for the preparation ofunit dosage forms for peroral administration are well known in the art.In some embodiments, tablets typically comprise conventionalpharmaceutically-compatible adjuvants as inert diluents, such as calciumcarbonate, sodium carbonate, mannitol, lactose and cellulose; binderssuch as starch, gelatin and sucrose; disintegrants such as starch,alginic acid and croscarmelose; lubricants such as magnesium stearate,stearic acid and talc. In one embodiment, glidants such as silicondioxide can be used to improve flow characteristics of thepowder-mixture. In one embodiment, coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. In some embodiments, theselection of carrier components depends on secondary considerations liketaste, cost, and shelf stability, which are not critical for thepurposes of this invention, and can be readily made by a person skilledin the art.

In one embodiment, the oral dosage form comprises predefined releaseprofile. In one embodiment, the oral dosage form of the presentinvention comprises an extended release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises a slow release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises an immediate release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form is formulatedaccording to the desired release profile of the pharmaceutical activeingredient as known to one skilled in the art.

Peroral compositions, in some embodiments, comprise liquid solutions,emulsions, suspensions, and the like. In some embodiments,pharmaceutically acceptable carriers suitable for preparation of suchcompositions are well known in the art. In some embodiments, liquid oralcompositions comprise from about 0.001% to about 0.933% of the desiredcompound or compounds, or in another embodiment, from about 0.01% toabout 10%.

In some embodiments, compositions for use in the methods of thisinvention comprise solutions or emulsions, which in some embodiments,are aqueous solutions or emulsions comprising a safe and effectiveamount of the compounds of the present invention and optionally, othercompounds, intended for topical intranasal administration. In someembodiments, the compositions comprise from about 0.001% to about 10.0%w/v of a subject compound, more preferably from about 00.1% to about2.0, which is used for systemic delivery of the compounds by theintranasal route.

In another embodiment, the pharmaceutical compositions are administeredby intravenous, intra-arterial, or intramuscular injection of a liquidpreparation. In some embodiments, liquid formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In oneembodiment, the pharmaceutical compositions are administeredintravenously, and are thus formulated in a form suitable forintravenous administration. In another embodiment, the pharmaceuticalcompositions are administered intra-arterially, and are thus formulatedin a form suitable for intra-arterial administration. In anotherembodiment, the pharmaceutical compositions are administeredintramuscularly, and are thus formulated in a form suitable forintramuscular administration.

Further, in another embodiment, the pharmaceutical compositions areadministered topically to body surfaces, and are thus formulated in aform suitable for topical administration. Suitable topical formulationsinclude gels, ointments, creams, lotions, drops and the like. Fortopical administration, the compounds of the present invention arecombined with an additional appropriate therapeutic agent or agents,prepared and applied as solutions, suspensions, or emulsions in aphysiologically acceptable diluent with or without a pharmaceuticalcarrier.

In one embodiment, pharmaceutical compositions of the present inventionare manufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

In one embodiment, pharmaceutical compositions for use in accordancewith the present invention is formulated in conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries, which facilitate processing of the active ingredientsinto preparations which can be used pharmaceutically. In one embodiment,formulation is dependent upon the route of administration chosen.

In one embodiment, injectables of the invention are formulated inaqueous solutions. In one embodiment, injectables of the invention areformulated in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiological salt buffer. In someembodiments, for transmucosal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art.

In one embodiment, the preparations described herein are formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. In some embodiments, formulations for injection are presentedin unit dosage form, e.g., in ampoules or in multidose containers withoptionally, an added preservative. In some embodiments, compositions aresuspensions, solutions or emulsions in oily or aqueous vehicles, andcontain formulatory agents such as suspending, stabilizing and/ordispersing agents.

The compositions also comprise, in some embodiments, preservatives, suchas benzalkonium chloride and thimerosal and the like; chelating agents,such as edetate sodium and others; buffers such as phosphate, citrateand acetate; tonicity agents such as sodium chloride, potassiumchloride, glycerin, mannitol and others; antioxidants such as ascorbicacid, acetylcystine, sodium metabisulfote and others; aromatic agents;viscosity adjustors, such as polymers, including cellulose andderivatives thereof; and polyvinyl alcohol and acid and bases to adjustthe pH of these aqueous compositions as needed. The compositions alsocomprise, in some embodiments, local anesthetics or other actives. Thecompositions can be used as sprays, mists, drops, and the like.

In some embodiments, pharmaceutical compositions for parenteraladministration include aqueous solutions of the active preparation inwater-soluble form. Additionally, suspensions of the active ingredients,in some embodiments, are prepared as appropriate oily or water-basedinjection suspensions. Suitable lipophilic solvents or vehicles include,in some embodiments, fatty oils such as sesame oil, or synthetic fattyacid esters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions contain, in some embodiments, substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. In another embodiment, the suspensionalso contains suitable stabilizers or agents which increase thesolubility of the active ingredients to allow for the preparation ofhighly concentrated solutions.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

In another embodiment, the pharmaceutical composition delivered in acontrolled release system is formulated for intravenous infusion,implantable osmotic pump, transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump is used (see Langer, supra;Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity tothe therapeutic target, i.e., the brain, thus requiring only a fractionof the systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990)).

In some embodiments, the active ingredient is in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-freewater-based solution, before use. Compositions are formulated, in someembodiments, for atomization and inhalation administration. In anotherembodiment, compositions are contained in a container with attachedatomizing means.

In one embodiment, the preparation of the present invention isformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

In some embodiments, pharmaceutical compositions suitable for use incontext of the present invention include compositions wherein the activeingredients are contained in an amount effective to achieve the intendedpurpose. In some embodiments, a therapeutically effective amount meansan amount of active ingredients effective to prevent, alleviate orameliorate symptoms of disease or prolong the survival of the subjectbeing treated.

In one embodiment, determination of a therapeutically effective amountis well within the capability of those skilled in the art.

Some examples of substances which can serve as pharmaceuticallyacceptable carriers or components thereof are sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose, and methyl cellulose; powdered tragacanth; malt;gelatin; talc; solid lubricants, such as stearic acid and magnesiumstearate; calcium sulfate; vegetable oils, such as peanut oil,cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma;polyols such as propylene glycol, glycerine, sorbitol, mannitol, andpolyethylene glycol; alginic acid; emulsifiers, such as the Tween™ brandemulsifiers; wetting agents, such sodium lauryl sulfate; coloringagents; flavoring agents; tableting agents, stabilizers; antioxidants;preservatives; pyrogen-free water; isotonic saline; and phosphate buffersolutions. The choice of a pharmaceutically acceptable carrier to beused in conjunction with the compound is basically determined by the waythe compound is to be administered. If the subject compound is to beinjected, in one embodiment, the pharmaceutically acceptable carrier issterile, physiological saline, with a blood-compatible suspending agent,the pH of which has been adjusted to about 7.4.

In addition, the compositions further comprise binders (e.g. acacia,cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropylcellulose, hydroxypropyl methyl cellulose, povidone), disintegratingagents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide,croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate),buffers (e.g., Tris-HCl., acetate, phosphate) of various pH and ionicstrength, additives such as albumin or gelatin to prevent absorption tosurfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acidsalts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate),permeation enhancers, solubilizing agents (e.g., glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite,butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose,hyroxypropylmethyl cellulose), viscosity increasing agents (e.g.carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum),sweeteners (e.g. aspartame, citric acid), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid,magnesium stearate, polyethylene glycol, sodium lauryl sulfate),flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethylphthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers orpoloxamines), coating and film forming agents (e.g. ethyl cellulose,acrylates, polymethacrylates) and/or adjuvants.

Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, cellulose (e.g. Avicel™, RC-591), tragacanth and sodiumalginate; typical wetting agents include lecithin and polyethylene oxidesorbitan (e.g. polysorbate 80). Typical preservatives include methylparaben and sodium benzoate. In another embodiment, peroral liquidcompositions also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

The compositions also include incorporation of the active material intoor onto particulate preparations of polymeric compounds such aspolylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts, or spheroplasts. Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance.

Also comprehended by the invention are particulate compositions coatedwith polymers (e.g. poloxamers or poloxamines) and the compound coupledto antibodies directed against tissue-specific receptors, ligands orantigens or coupled to ligands of tissue-specific receptors.

In some embodiments, compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. Inanother embodiment, the modified compounds exhibit substantially longerhalf-lives in blood following intravenous injection than do thecorresponding unmodified compounds. In one embodiment, modificationsalso increase the compound's solubility in aqueous solution, eliminateaggregation, enhance the physical and chemical stability of thecompound, and greatly reduce the immunogenicity and reactivity of thecompound. In another embodiment, the desired in vivo biological activityis achieved by the administration of such polymer-compound abducts lessfrequently or in lower doses than with the unmodified compound.

In some embodiments, preparation of an effective amount or dose can beestimated initially from in vitro assays. In one embodiment, a dose canbe formulated in animal models and such information can be used to moreaccurately determine useful doses in humans.

In one embodiment, toxicity and therapeutic efficacy of the activeingredients described herein can be determined by standardpharmaceutical procedures in vitro, in cell cultures or experimentalanimals. In one embodiment, the data obtained from these in vitro andcell culture assays and animal studies can be used in formulating arange of dosage for use in human. In one embodiment, the dosages varydepending upon the dosage form employed and the route of administrationutilized. In one embodiment, the exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. [See e.g., Fingl, et al., (1975) “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1].

In one embodiment, depending on the severity and responsiveness of thecondition to be treated, dosing can be of a single or a plurality ofadministrations, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

In one embodiment, the amount of a composition to be administered will,of course, be dependent on the subject being treated, the severity ofthe affliction, the manner of administration, the judgment of theprescribing physician, etc.

In one embodiment, compositions including the preparation of the presentinvention formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

In another embodiment, a cytokine as described herein is administeredvia systemic administration. In another embodiment, a cytokine asdescribed herein is administered by intravenous, intramuscular orsubcutaneous injection. In another embodiment, a cytokine as describedherein is in a lyophilized (i.e., freeze-dried) preparation incombination with complex organic excipients and stabilizers such asnonionic surface active agents (i.e., surfactants), various sugars,organic polyols and/or human serum albumin. In another embodiment, apharmaceutical composition comprises a lyophilized cytokine as describedin sterile water for injection. In another embodiment, a pharmaceuticalcomposition comprises a lyophilized cytokine as described in sterile PBSfor injection. In another embodiment, a pharmaceutical compositioncomprises a lyophilized cytokine as described in sterile 0.9% NaCl forinjection.

In another embodiment, the pharmaceutical composition comprises acytokine as described herein and complex carriers such as human serumalbumin, polyols, sugars, and anionic surface active stabilizing agents.See, for example, WO 89/10756 (Hara et al.—containing polyol andp-hydroxybenzoate). In another embodiment, the pharmaceuticalcomposition comprises a cytokine as described herein and lactobionicacid and an acetate/glycine buffer. In another embodiment, thepharmaceutical composition comprises a cytokine as described herein andamino acids, such as arginine or glutamate that increase the solubilityof interferon compositions in water. In another embodiment, thepharmaceutical composition comprises a lyophilized cytokine as describedherein and glycine or human serum albumin (HSA), a buffer (e.g. acetate)and an isotonic agent (e.g NaCl). In another embodiment, thepharmaceutical composition comprises a lyophilized cytokine as describedherein and phosphate buffer, glycine and HSA.

In another embodiment, the pharmaceutical composition comprising acytokine as described herein is stabilized when placed in bufferedsolutions having a pH of between about 4 and 7.2. In another embodiment,the pharmaceutical composition comprising a cytokine as described hereinis stabilized with an amino acid as a stabilizing agent and in somecases a salt (if the amino acid does not contain a charged side chain).

In another embodiment, the pharmaceutical composition comprising acytokine as described herein is a liquid composition comprising astabilizing agent at between about 0.3% and 5% by weight which is anamino acid.

In another embodiment, the pharmaceutical composition comprising acytokine as described herein provides dosing accuracy and productsafety. In another embodiment, the pharmaceutical composition comprisinga cytokine as described herein provides a biologically active, stableliquid formulation for use in injectable applications. In anotherembodiment, the pharmaceutical composition comprises a non-lyophilizedcytokine as described herein.

In another embodiment, the pharmaceutical composition comprising acytokine as described herein provides a liquid formulation permittingstorage for a long period of time in a liquid state facilitating storageand shipping prior to administration.

In another embodiment, the pharmaceutical composition comprising acytokine as described herein comprises solid lipids as matrix material.In another embodiment, the injectable pharmaceutical compositioncomprising a cytokine as described herein comprises solid lipids asmatrix material. In another embodiment, the production of lipidmicroparticles by spray congealing was described by Speiser (Speiser andal., Pharm. Res. 8 (1991) 47-54) followed by lipid nanopellets forperoral administration (Speiser EP 0167825 (1990)). In anotherembodiment, lipids which are used are well tolerated by the body (e.g.glycerides composed of fatty acids which are present in the emulsionsfor parenteral nutrition).

In another embodiment, the pharmaceutical composition comprising acytokine as described herein is in the form of liposomes (J. E.Diederichs and al., Pharm./nd. 56 (1994) 267-275).

In another embodiment, the pharmaceutical composition comprising acytokine as described herein comprises polymeric microparticles. Inanother embodiment, the injectable pharmaceutical composition comprisinga cytokine as described herein comprises polymeric microparticles. Inanother embodiment, the pharmaceutical composition comprising a cytokineas described herein comprises nanoparticles. In another embodiment, thepharmaceutical composition comprising a cytokine as described hereincomprises liposomes. In another embodiment, the pharmaceuticalcomposition comprising a cytokine as described herein comprises lipidemulsion. In another embodiment, the pharmaceutical compositioncomprising a cytokine as described herein comprises microspheres. Inanother embodiment, the pharmaceutical composition comprising a cytokineas described herein comprises lipid nanoparticles. In anotherembodiment, the pharmaceutical composition comprising a cytokine asdescribed herein comprises lipid nanoparticles comprising amphiphiliclipids. In another embodiment, the pharmaceutical composition comprisinga cytokine as described herein comprises lipid nanoparticles comprisinga drug, a lipid matrix and a surfactant. In another embodiment, thelipid matrix has a monoglyceride content which is at least 50% w/w.

In one embodiment, compositions of the present invention are presentedin a pack or dispenser device, such as an FDA approved kit, whichcontain one or more unit dosage forms containing the active ingredient.In one embodiment, the pack, for example, comprise metal or plasticfoil, such as a blister pack. In one embodiment, the pack or dispenserdevice is accompanied by instructions for administration. In oneembodiment, the pack or dispenser is accommodated by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsfor human or veterinary administration. Such notice, in one embodiment,is labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert.

In one embodiment, it will be appreciated that the cytokines of thepresent invention can be provided to the individual with additionalactive agents to achieve an improved therapeutic effect as compared totreatment with each agent by itself. In another embodiment, measures(e.g., dosing and selection of the complementary agent) are taken tominimize adverse side effects which are associated with combinationtherapies.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A Laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8^(th) Edition), Appleton &Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “SelectedMethods in Cellular Immunology”, W. H. Freeman and Co., New York (1980);available immunoassays are extensively described in the patent andscientific literature, see, for example, U.S. Pat. Nos. 3,791,932;3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262;3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876;4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M.J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and HigginsS. J., eds. (1985); “Transcription and Translation” Hames, B. D., andHiggins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed.(1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A PracticalGuide to Molecular Cloning” Perbal, B., (1984) and “Methods inEnzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide ToMethods And Applications”, Academic Press, San Diego, Calif. (1990);Marshak et al., “Strategies for Protein Purification andCharacterization—A Laboratory Course Manual” CSHL Press (1996); all ofwhich are incorporated by reference. Other general references areprovided throughout this document.

Example 1 Generation of EPO Constructs Materials and Methods:

Construction of expression vector pa-dhfr: pCI-neo mammalian expressionvector was purchased from Promega (Catalog No. E1841). The vectorcontains a CMV IE enhancer/promoter and neomycin phosphotransferasegene. The pSV2-dhfr clone was purchased from ATCC (Catalog No. 37146).The plasmid contains the murine dhfr gene. The construction of pCI-dhfrvector was performed as follows:

The pSV2-dhfr plasmid was digested with restriction enzyme BglII (3′ endof the dhfr gene). DNA polymerase I, Large (Klenow) Fragment was used tofill in the 5′ overhangs to form blunt ends. The DNA was then digestedwith restriction enzyme AvrII (5′ end of the dhfr gene). The dhfr gene(AvrII-blunt end) fragment was isolated.

The pCI-neo vector was digested with restriction enzyme BstXI (3′ end ofthe neo gene). DNA polymerase I, Large (Klenow) Fragment was used toremove the 3′ overhangs to form blunt ends. The DNA was then digestedwith restriction enzyme AvrII (5′ end of the neo gene). The expressionvector (AvrII-blunt end) was isolated.

The dhfr gene was ligated into pCI vector to form an expression vectorcontaining the dhfr gene (pCI-dhfr).

Construction of hEPO-CTP variants: A cassette gene containing theC-Terminal peptide (CTP) of the beta subunit of hCG was fused to thecoding sequence of human EPO (NP_(—)000790.2) at different locations.Four EPO-CTP variants were constructed as illustrated in FIGS. 1A-D. TheproEPO signal peptide was used for the construction of the secretedEPO-CTP variants. XbaI-NotI fragments containing Epo sequences wereligated into the pCI-dhfr expression vector of the present invention.

Table 2 hereinbelow summarizes the primer sequences used forconstructing the CTP-containing polypeptides of the present invention.

TABLE 2 SEQ Restriction site Primer ID (underlined in number NO sequencesequence)  1  7 5′ AATCTAGAGGTCATCATGGGGGTGC 3′ XbaI  2  85′ATTGCGGCCGCGGATCCAGAAGACCTTTATTG 3′ NotI 17^(R)  9 5′TAAATATTGGGGTGTCCGAGGGCCC 3′ SspI 10 10 5′CCAATATTACCACAAGCCCCACCACGCCTCAT 3′ SspI 11^(R) 115′TGCGGCCGCGGATCCTTATCTGTCCCCTGTCCTGC 3′ NotI 15 12 5′GCCCTGCTGTCGGAAGC 3′  2^(R) 13 5′ ATTGCGGCCGCGGATCCAGAAGACCTTTATTG NotI23^(R) 14 5′CTTTGAGGAAGAGGAGCCCAGGACTGGGAGGC3′ 24 15 5′CCTGGGCTCCTCTTCCTCAAAGGC 3′ 38^(R) 16 5′ GCTTCCGACAGCAGGGC 3′

EPO-1 701-1-p17-6 (Epo-1—SEQ ID NO: 1): The XbaI-NotI 702 bp fragmentwas constructed by PCR using the above primers (SEQ ID NOs: 7-16). Thenthe XbaI-NotI PCR fragment containing Epo-ctp sequence was ligated intopCI-dhfr expression vector.

EPO-2 701-2-p24-2 (Epo-2—SEQ ID NO: 2): The XbaI/ApaI fragment (hGH-ctp)of pCI-dhfr-401-2-p21-2 (hGH-ctpx2) was replaced by the XbaI/ApaIfragment (EPO-ctp) of 701-1-p17-6 to create an Epo-ctpx2.

EPO-4-701-4-p42-1 (Epo-4—SEQ ID NO: 4): First, a fragment frompCI-dhfr-EPO-ctp (701-1-p17-6) was constructed by PCR using primers 1and 17 followed by XbaI/SspI to digestion. This resulted in a fragmentcontaining EPO and partial 5′ CTP.

Secondly, a new fragment was constructed by overlapping PCR, onpGT123-hEpo as a template, using primer 10 and primer 11. SspI/NotIdigestion resulted in a fragment containing 3′ partial CTP and Epo.

The two fragments were ligated into pCI-dhfr to construct thep701-4-p42-1 clone.

EPO-3-p56-6 (Epo-3—SEQ ID NO: 3): Primers were purchased fromSigma-Genosys. A PCR reaction was performed using primer 15 (SEQ ID NO:12) and primer 2^(R) (SEQ ID NO: 13) and plasmid DNA of pCI-dhfr-EPO-ctpx2 (701-2-p24-2) as a template. As a result of the PCR amplification, a486 bp product was formed and ligated into TA cloning vector(Invitrogen, catalog K2000-01). Stu I-NotI fragment containing *Epo-ctpx2 sequence was isolated (209 bp).

Three sequential PCR reactions were performed. The first reaction wasconducted with primer 1 (SEQ ID NO: 7) and primer 23^(R) (SEQ ID NO: 14)and plasmid DNA of pGT123-epo-ctp as a template; as a result of the PCRamplification, an 80 bp product was formed (signal peptide).

The second reaction was conducted with primer 24 (SEQ ID NO: 15) andprimer 11^(R) (SEQ ID NO: 11) and plasmid DNA of 701-4-p42-1 as atemplate; as a result of the PCR amplification, a 610 bp product wasformed.

The last reaction was conducted with primers 1 (SEQ ID NO: 7) and 11^(R)(SEQ ID NO: 11) and a mixture of the products of the previous tworeactions as a template; as a result of the PCR amplification, a 700 bpproduct was formed and the XbaI-StuI fragment was isolated.

The two fragments (XbaI-StuI and StuI-NotI) were inserted into theeukaryotic expression vector pCI-dhfr (triple ligation) to yield the701-3-p56-6 clone.

EPO-5-p91-4 (Epo-5—SEQ ID NO: 5—(ctp-Epo)): Primers were ordered fromSigma-Genosys. A PCR reaction was performed using primer 1 (SEQ ID NO:7) and primer 11^(R) (SEQ ID NO: 11) and plasmid DNA ofpCI-dhfr-ctp-EPO-ctp x2 (701-3-p56-6) as a template; as a result of thePCR amplification, a 670 bp product was formed and ligated into TAcloning vector (Invitrogen, catalog K2000-01). XbaI-NotI fragmentcontaining ctp-Epo sequence was ligated into the eukaryotic expressionvector pCI-dhfr to yield the 701-5-p91-4 clone.

EPO-6-p90-1 (Epo-6—SEQ ID NO: 6—(ctp-Epo-ctp)): Three PCR reactions wereperformed. The first reaction was conducted with primer 1 (SEQ ID NO: 7)and primer 38^(R (SEQ ID NO:) 16) and plasmid DNA of 701-3-p56-6 as atemplate; as a result of the PCR amplification, a 400 bp product wasformed.

The second reaction was conducted with primer 15 (SEQ ID NO: 12) andprimer 2^(R) (SEQ ID NO: 13) and plasmid DNA of 701-1-p17-6 as atemplate; as a result of the PCR amplification, a 390 bp product wasformed.

The last reaction was conducted with primers 1 (SEQ ID NO: 7) and 2^(R)(SEQ ID NO: 13) and a mixture of the products of the previous tworeactions as a template; as a result of the PCR amplification, a 787 bpproduct was formed and ligated into a TA cloning vector (Invitrogen,catalog K2000-01). The XbaI-NotI fragment containing ctp-Epo-ctpsequence was ligated into the eukaryotic expression vector pCI-dhfr toyield the 701-6-p90-1 clone.

Example 2 Expression and Isolation of EPO-CTP Polypeptides Materials andMethods

DNA transfection and clone selection: DG44 cells were transfected withpCI-DHFR expression vectors containing EPO-CTP variants using FuGENE6Reagent (FuGENE Transfection Reagent—Roche Cat.11 815 091 001). 48 hrfollowing transfection, cells were diluted and seeded at 50-200 cellsper well in a selective medium (CD DG44 Medium w/o HT (Gibco: Scotlandpart: #07990111A) Sku num.:ME060027 supplemented with 8 mM L-Glutamine(Biological Industries: Cat: 03-020-1A) and 18 mL/L of 10% Pluronic F-68solution (Gibco: Cat: 240040-032). Selected clones were screened forhighest protein production using commercial ELISA. Three to fiveproducing clones per each variant were frozen for a backup cell bank. Aselected clone for each variant was adapted to growth in larger scalecultures up to 1 L flasks on an orbital shaker platform. Supernatantswere collected and analyzed by ELISA, SDS-PAGE and Western blot.Following the withdrawal of aliquots, the protein-containingsupernatants were kept frozen until further use.

Cell culture: DG44 cells were maintained in DG44 medium with HT (cat#12610-010, Gibco) supplemented with 8 mM L-Glutamine (BiologicalIndustries: Cat: 03-020-1A) and 18 mL/L of 10% Pluronic F-68 solution(Gibco: Cat: 240040-032), at 37° C. in a humidified 8% CO₂ incubator.Transfected clones were maintained in DG44 basal medium without HTsupplement, hypoxanthine and thymidine, with pluronic acid andL-glutamine.

Sample preparation: Supernatants were collected, filtrated and analyzedby ELISA to determine protein concentration. SDS-PAGE and Western blotwere used to determine purity and identity. Following ELISA, sampleconcentrations were defined and the solution was dialyzed against PBS.Following the withdrawal of aliquots, the protein-contained supernatantswere kept frozen at −20° C. until further use.

Western Blotting: Samples were electrophoresed on nondenaturing 15SDS-polyacrylamide gels. Gels were allowed to equilibrate for 10 min in25 mM Tris and 192 mM glycine in 20% (vol/vol) methanol). Proteins weretransferred to a 0.2 μm pore size nitrocellulose membrane (Sigma, SaintLouis, Mo.) at 250 mA for 3 h, using a Mini Trans-Blot electrophoresiscell (Biorad Laboratories, Richmond, Calif.). The nitrocellulosemembrane was incubated in 5% non-fat dry milk for 2 h at roomtemperature. The membrane was incubated with EPO anti-serum (1:1000titer) overnight at 4° C. followed by three consecutive washes in PBScontaining 0.1% Tween (10 min/wash). The membrane was incubated withsecondary antibody conjugated to Horse Radish Peroxidase (HRP) (Zymed,San Francisco, Calif.) for 2 h at room temperature, followed by threewashes. Finally, the nitrocellulose paper was reacted with enhancedchemiluminescent substrate (ECL) (Pierce, Rockford, Ill.) for 5 min,dried with a Whatman sheet, and exposed to X-ray film.

Results

Table 3 hereinbelow shows the concentrations of the various CTP-modifiedEPO forms obtained from 5 selected clones and their preparation forfurther testing.

TABLE 3 Post dilution in Stock Mock sup. According Post ultra- Titer toEpo3 titer filtration #Version # Clone IU/ml¹ IU/ml² IU/ml³ Epo0 17 3093102 335 SEQ ID NO: 16 Epo1 47 1049 104 291 SEQ ID NO: 1 Epo2 67 2160 110303 SEQ ID NO: 2 Epo3 85 105 119 392 SEQ ID NO: 3 Epo4 112 6100 ND 342SEQ ID NO: 4

-   -   1. EPO variants stock concentration were determined by ELISA        (Quantikine IVD Epo ELISA, DEPOO, R&D Systems).    -   2. Samples EPO-0, 1, 2 and 4 were diluted to 105 IU/ml in mock        sup (Adjusted to Epo3 titer). Epo-0=wild type EPO expressed in        the same system as the CTP-modified EPOs.    -   3. All samples were concentrated and dialyzed by ultrafiltration        against PBS to a final concentration of 180 IU/ml.

All proteins were detected by Western blot as illustrated in FIG. 2.

Example 3 Biological Activity of the EPO-CTP Polypeptides of the PresentInvention

The TF-1 bioactivity test represents the ability of the EPO-CTP variantto bind its receptor and then stimulate activity which results in cellproliferation. Therefore, this test was used as a first step inevaluating the biological potency of the EPO-CTP polypeptides of thepresent invention.

Materials and Methods

Cell Proliferation Analysis: Proliferation assay was performed with thecell line TF-1, measuring levels of MTT cellular stain as a function ofEPO activity (Kitamura et al., Kitamura, T. et al. (1989) Establishmentand characterization of a unique human cell line that proliferates;Hammerling U. et al. In vitro bioassay for human erythropoietin based onproliferative stimulation of an erythroid cell line and analysis ofcarbohydrate-dependent microheterogeneity. Journal of Pharm. Biomed.Analysis 14(11): 1455-1469 (1996)). Exponentially growing TF-1 cellswere washed twice, plated at about 10⁴ cells/well in microtiter plates,and incubated in basal medium with a titrated dilution series of EPO(Recormon®), EPO standard (NIBSC standard), rhEPO (MOD-7010), MOD-701variants (EPO-1, EPO-2, EPO-3 and EPO-4) for 48 hours. Four hours priorto assaying for cell proliferation, MIT reagent was added to the wells,and absorbance was measured by ELISA reader. A calculated proteinconcentration value for each variant protein was obtained from Eprex®s(Epoetin (EPO)-man-made form of the human hormone) dose-responsestandard curve.

Results

The in vitro biological activity of EPO polypeptides was determined withan Epo-dependent cell line, human erythroleukemia TF-1 (DSMZ Cell Bank)[Dong et al., Biochemical and Biophysical Research Communications,Volume 339, Issue 1, 6 Jan. 2006, Pages 380-385]. The MTT assay wasperformed [Hammerling U. et al. In vitro bioassay for humanerythropoietin based on proliferative stimulation of an erythroid cellline and analysis of carbohydrate-dependent microheterogeneity. Journalof Pharm. Biomed. Analysis 14(11): 1455-1469 (1996)], and the laboratorystandard of EPO used to generate the standard curve was calibratedagainst the International Standard (Epo ampoule code 87/684 of NIB S C).

The results are summarized in Table 4 hereinbelow. The results indicatethat the highest potency was achieved by using EPO 3 and EPO 0 in both 2and 0.5 IU/ml concentrations.

TABLE 4 TF-1 Bioactivity IU/ml Eprex STD EPO 0 EPO 1 EPO 2 EPO 3 EPO 4IU/ml SEQ ID NO: 16 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4Recormon ® EPO st 2 4.93 2.32 2.13 6.91 3.55 3.44 7.40 0.5 1.60 0.760.53 1.34 0.84 0.87 1.53

Conclusion

As summarized in Table 4 hereinabove, different levels of potency wereexerted by EPO-CTP polypeptides, indicating differences in receptorbinding. EPO-CTP polypeptides differ by the number of CTP cassettes andthe location to which they are fused. EPO-1 and EPO-2 contain 1 CTPsequence or 2 CTP sequences at the C-terminal of EPO, while EPO-3contains 1 CTP at the N-terminal and 2 CTP sequences at the C-terminal.EPO-4 is a dimer of two EPO molecules linked by CTP sequence. EPO-3demonstrated a potency level quite similar to WT-EPO, while EPO-1 andEPO-4 were about 50% less potent than WT-EPO, and EPO-2 potency was evenless than 50%.

Example 4 Evaluation of the EPO-CTP Polypeptides of the PresentInvention in a Mouse Model

The following experiment was performed in order to compare thebio-activity of the EPO-CTP polypeptides of the present invention andcommercial EPO.

Materials and Methods Animals:

Species/Strain: ICR or CD-1 Mice of either sex about 20-25 gGroup Size: n=7No. Groups: 9Total No. Animals: n=63Experimental design of the study: The experiment was set up assummarized in Table 5 hereinbelow.

TABLE 5 Group No. Mice TREATMENT Dosing No. per Group Compound DoseLevel Regimen 1 n = 7 Vehicle 0 2 MOCK 3 MOD-7010 15 μg/kg 4 MOD-7011 5MOD-7012 6 MOD-7013 7 MOD-7014 8 Commercial 15 μg/kg 9 rhEPO  5 μg/kg 3× weekly

Animal treatment: All animals were administered with either control orthe test EPO polypeptides of the present invention by bolus injection.The injection volume did not exceed 10 ml/kg. The length of theexperiment was 22 days. A morbidity and mortality check was performeddaily.

Reticulocyte count and hematocrit (hct) examination: Reticulocyte countwas carried out in all test animals at day 2 and 14 hrs following the1^(st) respective vehicle or treatment injection. HCT was determined inall animals once prior to initial treatment (“0” Baseline control) andat 24 hrs after the 1^(st) respective vehicle or treatment injection,and thereafter twice weekly until study termination (Day 22).

Results

The hematocrit results which are illustrated in FIGS. 3-5 show that EPO3 has the highest hematocrit percentage change from baseline compared toEPO 1, EPO 2, Recormon® 1, Recormon® 3, rhEPO, and vehicle. The resultsdemonstrating the percentage of reticulocytes in mice treated with theEPO-CTP polypeptides are summarized in Table 6 hereinbelow. Theseresults show that EPO-3 is the most potent stimulator of erythropoiesis.

TABLE 6 % reticulocytes Days 2 14 Control 3.72 3.46 1.08 0.8 Mock 3.53.64 0.6 1.13 7010 SEQ ID NO: 16 3.5 3.9 0.6 1.54 7011 SEQ ID NO: 1 3.521.94 1.38 1.08 7012 SEQ ID NO: 2 3.82 3.0 1.02 0.88 7013 SEQ ID NO: 32.66 5.20 0.97 2.96 7014 SEQ ID NO: 4 3.48 3.82 0.71 0.90 Recormon ® 1/W3.23 3.27 0.73 0.59 Recormon ® 3/w 4.13 4.24 1.21 1.14

Conclusion

The in vivo experiment was designed to measure two parameters; the firstwas to measure erythropoiesis parameters such as percentage ofreticulocytes and increase of hemoglobin, RBC and hematocrit levels. Thesecond was to measure the durability of the biological activity of eachvariant by injecting once weekly doses.

A superior performance of EPO-3 in its ability to stimulateerythropoiesis was observed in normal mice.

Example 5 Comparison of the EPO-CTP Polypeptides of the PresentInvention to Aranesp®

The following experiment was performed in order to compare thebiological activity of a single bolus dose of some EPO-CTP polypeptidesof the present invention, commercial EPO and Aranesp®. Aranesp® is acommercial long-acting recombinant erythropoietin in which two sitemutations were introduced, resulting in two additional N-glycosylationsites and an increase in the number of incorporated sialic acidresidues.

Materials and Methods Animals:

Species/Strain: Female CD-1 Mice of either sex about 20-25 gGroup Size: n=3Experimental design of the study: The experiment was set up assummarized in Table 7 hereinbelow.

TABLE 7 animals/ Dose Dose Group group/ Solution Conc. VolumeTime-Points * # Test Article time-point (μg/mL) (mL/kg) (hourspost-administration) 1 MOD-7010 3 1.5 10 0 (Pre-dose), 0.25, 0.5, 1, 2,6, SEQ ID NO: 11 24, 48, 96, 168, 216, 264 and 336 hr post-doseadministration 2 MOD-7013 3 1.5 10 0.25, 0.5, 1, 2, 6, 24, 48, 96, SEQID NO: 3 168, 216, 264 and 336 hr post- dose administration 3 Aranesp ®3 1.5 10 0.25, 0.5, 1, 2, 6, 24, 48, 96, 168, 216, 264 and 336 hr post-dose administration

Animal treatment: All animals were administered with either control orthe test EPO polypeptides of the present invention by bolus injection.The injection volume did not exceed 10 ml/kg. The length of theexperiment was 14 days. A morbidity and mortality check was performeddaily.

Reticulocyte count and hematocrit (hct) examination: Reticulocyte countand hematocrit examination were performed as described above.

Results

The results are illustrated in FIGS. 6-9. Following a single. i.v.injection of 15 μg/kg of EPO 3, all three blood parameters associatedwith erythropoietin i.e. number of reticulocytes, hemoglobin level andhematocrit, were improved relative to those obtained with similarinjected dose of rhEPO or Aranesp®.

Example 6 Comparison of the Pharmacokinetics of EPO-CTP Polypeptides ofthe Present Invention to Aranesp®

The following experiment was performed in order to compare thepharmacokinetics of EPO-CTP polypeptide of the present invention,commercial EPO and Aranesp®.

Materials and Methods

Serum samples were analyzed in order to determine specific concentrationlevels for each sample. Concentration and time-point data were processedusing WinNonLin noncompartmental analysis. Parameters determinedincluded: AUC, CL, Ke, t_(1/2), C_(max), T_(max), and Vdz.

Serum concentrations were determined using two ELISA kits in parallel.EPO-3 serum concentration was measured using StemCell ELISA kit incomparison to EPO-0 and Aranesp® serum concentration which weredetermined using R&D system ELISA kit.

Results

The results of the pharmacokinetic analysis are summarized in Table 8,hereinbelow. These results show that EPO 3 exhibited favorablepharmacokinetic measures as indicated for example in AUC measures,t_(1/2), and C_(max). T_(max) measures were equal to EPO-0, EPO-3, andAranesp®.

TABLE 8 Parameters Units EPO-0 EPO-3 Aranesp ® AUClast hr*mIU/mL 31739306072 178661 CL{circumflex over ( )} mL/hr/kg 1.1152 0.2188 0.1207 Ke1/hr 0.157 0.0529 0.0639 t_(1/2) hr 4.4139 13.1141 10.84 Cmax mIU/mL10766 16466 13266 Tmax Hr 0.25 0.25 0.25 Vdz mL/kg 7.1017 4.1394 1.8877

The results of the serum concentration analysis are illustrated in FIG.9. These results show that EPO-3 was still detectable in the serum afterabout 190 hours. Both EPO-0 and Aranesp® were not detectable in theserum after about 140 hours and 50 hours, respectively.

Conclusion

Clearance of EPO-3 (MOD-7013) from the blood of CD-1 mice wassignificantly slower than that for rhEPO or Aranesp®. The correspondingcalculated half life times were: rhEPO—4.41 h; Aranesp®—0.84 h; andMOD-7013—13.11 h.

Example 7 Generation of hGH Constructs Materials and Methods

Four hGH clones (variants of 20 kD protein) were synthesized. Xba I-NotI fragments containing hGH sequences from the four variants were ligatedinto the eukaryotic expression vector pCI-dhfr previously digested withXbaI-NotI. DNA from the 4 clones (401-0, 1, 2, 3 and 4) was prepared.Another partial hGH clone (1-242 bp) from 22 kD protein was alsosynthesized (0606114). Primers were ordered from Sigma-Genosys. Theprimer sequences used to generate the hGH-CTP polypeptides of thepresent invention are summarized in Table 9, hereinbelow.

TABLE 9 SEQ Restriction site Primer ID (underlined in number NO sequencesequence) 25 27 5′ CTCTAGAGGACATGGCCAC 3′ XbaI 32^(R) 28 5′ACAGGGAGGTCTGGGGGTTCTGCA 3′ 33 29 5′ TGCAGAACCCCCAGACCTCCCTGTGC 3′ 4^(R) 30 5′ CCAAACTCATCAATGTATCTTA 3′ 25 31 5′ CTCTAGAGGACATGGCCAC 3′XbaI 35^(R) 32 5′ CGAACTCCTGGTAGGTGTCAAAGGC 3′ 34 33 5′GCCTTTGACACCTACCAGGAGTTCG 3′ 37^(R) 34 5′ACGCGGCCGCATCCAGACCTTCATCACTGAGGC 3′ NotI 39^(R) 35 5′GCGGCCGCGGACTCATCAGAAGCCGCAGCTGCCC 3′

Construction of 402-O-p69-1 (hGH) SEQ ID NO: 36: MOD-4020 is the wildtype recombinant human growth hormone (without CTP) which was preparedfor use as control in the below described experiments.

Three PCR reactions were performed. The first reaction was conductedwith primer 25 and primer 32^(R) and plasmid DNA of 0606114 (partialclone of hGH 1-242 bp) as a template; as a result of the PCRamplification, a 245 bp product was formed.

The second reaction was conducted with primer 33 and primer 4^(R) andplasmid DNA of 401-O-p57-2 as a template; as a result of the PCRamplification, a 542 bp product was formed.

The last reaction was conducted with primers 25 and 4^(R) and a mixtureof the products of the previous two reactions as a template; as a resultof the PCR amplification, a 705 bp product was formed and ligated intothe TA cloning vector (Invitrogen, catalog K2000-01). The XbaI-NotIfragment containing hGH-0 sequence was ligated into the eukaryoticexpression vector pCI-dhfr. The vector was transfected into DG-44 CHOcells. Cells were grown in protein-free medium.

Construction of 402-1-p83-5 (hGH-CTP)—SEQ ID NO: 37 and402-2-p72-3(hGH-CTPx2)—SEQ ID NO: 38: MOD-4021 is a recombinant humangrowth hormone which was fused to 1 copy of the C-terminal peptide ofthe beta chain of human Chorionic Gonadotropin (CTP). The CTP cassetteof MOD-4021 was attached to the C-terminus (one cassette). MOD-4022 is arecombinant human growth hormone which was fused to 2 copies of theC-terminal peptide of the beta chain of human Chorionic Gonadotropin(CTP). The two CTP cassettes of MOD-4022 were attached to the C-terminus(two cassettes).

Construction of hGH-CTP and hGH-CTP-CTP was performed in the same way asthe construction of hGH-0. pCI-dhfr-401-1-p20-1 (hGH*-ctp) andpCI-dhfr-401-2-p21-2 (hGH*-ctp x2) were used as templates in the secondPCR reaction.

MOD-4021 and MOD-4022 were expressed in DG-44 CHO cells. Cells weregrown in protein-free medium. The molecular weight of MOD-4021 is ˜30.5Kd since hGH has a MW of 22 Kd, while each “CTP cassette” contributes8.5 Kd to the overall molecular weight (see FIG. 10). The molecularweight of MOD-4022 is ˜39 Kd (see FIG. 10).

Construction of 402-3-p8′-4 (CTP-hGH-CTP-CTP)—SEQ ID NO: 39 and402-4-p82-9(CTP*hGH-CTP-CTP)—SEQ ID NO: 40: MOD-4023 is a recombinanthuman growth hormone which was fused to 3 copies of the C-terminalpeptide of the beta chain of human Chorionic Gonadotropin (CTP). Thethree CTP cassettes of MOD-4023 were attached to both N-terminus (onecassette) and the C-terminus (two cassettes). MOD-4024 is a recombinanthuman growth hormone which is fused to 1 truncated and 2 complete copiesof the C-terminal peptide of the beta chain of human ChorionicGonadotropin (CTP). The truncated CTP cassette of MOD-4024 was attachedto the N-terminus and two CTP cassettes were attached to the C-terminus(two cassettes).

Three PCR reactions were performed. The first reaction was conductedwith primer 25 and primer 35^(R) and plasmid DNA of p401-3-p12-5 or401-4-p22-1 as a template; as a result of the PCR amplification, a 265or 220 bp product was formed. The second reaction was conducted withprimer 34 and primer 37^(R) and plasmid DNA of TA-hGH-2-q65-1 as atemplate; as a result of the PCR amplification, a 695 bp product wasformed. The last reaction was conducted with primers 25 and 37^(R) and amixture of the products of the previous two reactions as a template; asa result of the PCR amplification, a 938 or 891 bp product was formedand ligated into TA cloning vector (Invitrogen, catalog K2000-01). XbaI-Not I fragment containing hGH sequence was ligated into our eukaryoticexpression vector pCI-dhfr.

MOD-4023 and MOD-4024 were expressed in DG-44 CHO cells. Cells weregrown in protein-free medium. The molecular weight of MOD-4023 is ˜47.5Kd (see FIG. 10) and the molecular weight of MOD-4024 is ˜43.25 Kd (seeFIG. 10).

Construction of 402-6-p95α-8 (CTP-hGH-CTP)—SEQ ID NO: 41: Constructionof hGH-6 was performed in the same way as the construction of hGH-3.pCI-dhfr-402-1-p83-5 (hGH-ctp) was used as a template in the second PCRreaction.

Construction of 402-0.5-p96-4 (CTP-hGH)—SEQ ID NO: 42: PCR reaction wasperformed using primer 25 and primer 39^(R) and plasmid DNA ofpCI-dhfr-ctp-EPO-ctp (402-6-p95a-8) as a template; as a result of thePCR amplification, a 763 bp product was formed and ligated into TAcloning vector (Invitrogen, catalog K2000-01). Xba I-Not I fragmentcontaining ctp-hGH sequence was ligated into our eukaryotic expressionvector pCI-dhfr to yield the 402-5-p96-4 clone.

Example 8 In Vivo Bioactivity Tests of hGH-CTP Polypeptides of thePresent Invention

The following experiment was performed in order to test the potentiallong acting biological activity of hGH-CTP polypeptides in comparisonwith commercial recombinant human GH and MOD-4020.

Materials and Methods

Female hypophysectomized rats (60-100 g) received a weekly s.c.injection of 21.7 μg hGH-CTP polypeptides or a once daily 5 μg s.c.injection of control commercial rhGH.

Weight was measured in all animals before treatment, 24 hours followingfirst injection and then every other day until the end of the study onday 21. Each point represents the group's average weight gain percentage((Weight day 0-weight last day)/Weight day 0). Average weight gain wasnormalized against once-daily injection of commercial hGH. The treatmentschedule is summarized in Table 10.

TABLE 10 Equi- Accumu- Treat- molar late Dose ment Dose Dosage Vol. No.Drug N Route Schedule (μg/rat) (μg/rat) (ml) 1 Vehicle 7 s.c. days 1, 7NA NA 0.25 and 13; 1/W 2 Mock 7 s.c. days 1, 7 NA NA 0.25 and 13; 1/W 3MOD-4020 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID and 13; NO: 36 1/W 4MOD-4021 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID and 13; NO: 37 1/W 5MOD-4022 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID and 13; NO: 38 1/W 6MOD-4023 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID and 13; NO: 39 1/W 7MOD-4024 7 s.c. days 1, 7 21.7 65 0.25 SEQ ID and 13; NO: 40 1/W 8Commercial 7 s.c. days 1, 7 21.7 65 0.25 hGH v.1 and 13; 1/W 9Commercial 7 s.c. days 1-13; 5 65 0.25 hGH v.1 d/W

Results

Results are summarized in FIG. 11. These results show that MOD-4023 (SEQID NO: 39) and MOD-4024 (SEQ ID NO: 40) induced over 120% weight gaincompared to commercial rhGH which induced 100% weight gain.

Conclusion

Three weekly doses (Days of injections;1, 7, and 13) of 21.7 μg ofMOD-4023 (SEQ ID NO: 39) and MOD-4024 (SEQ ID NO: 40) induced a 30%greater weight increase in hypophysectomised rats compared to commercialrhGH injected at the same accumulated dose which was administered onceper day at a dose of 5 m for 13 days.

Example 9 The Superiority of hGH-CTP Polypeptides of the PresentInvention

Pharmacokinetic studies

Single-dose pharmacokinetic studies were conducted in Sprague-Dawleyrats. All animal experimentation was conducted in accordance with theAnimal Welfare Act, the Guide for the Care and Use of LaboratoryAnimals, and under the supervision and approval of the InstitutionalAnimal Care and Use Committees of Modigene Biotechnology General Ltd.Rats were housed either individually or two per cage in rooms with a12-h light/dark cycle. Access to water (municipal supply) andnoncertified rodent chow was provided ad libitum.

To compare the pharmacokinetics of MOD-4023 and GH in rats, four groupsof Sprague-Dawley rats (270-290 g), three to six male rats per group.The rats were randomly assigned to four treatment groups (see Table 11).Rats were administered a single s.c. or i.v. injection of GH (50 μg/kgi.v. or s.c.) or MOD-4023 (108 μg/kg i.v. or s.c.). With the exceptionof the predose sample, which was collected under isoflurane anesthesia,blood collection was performed in unanesthetized animals. Blood samples(approximately 0.25 ml) were collected in EDTA-coated microtainers forELISA analyses of MOD-4023 plasma concentration at the times outlined inTable 11. After each sampling, the blood volume was replaced with anequal volume of sterile 0.9% saline. Samples were stored on wet ice forup to 1 h prior to centrifugation and plasma harvest. Plasma sampleswere stored at approximately −20° C. prior to analysis.

TABLE 11 Experimental design of rat pharmacokinetic study No. ofConcentration animals/ Dose Injected (μg/ml)/ Trt. Test group/ DoseLevel Vol. Total vol. Time-Points * Grp. Article timepoint Route Gender(μg/kg) (μl) (ml) (hours post-dose) 1 Biotropin ® 6# s.c. Male 50 500 20/5 0 (Pre-dose) 0.5, 2, 4, 8, 24, 48 2 MOD-4023 6# s.c. Male 108 50043.2/5 0.5, 2, 4, 8, 24, 48, 72, 96 3 Biotropin ® 6# i.v. Male 50 300 20/3 0, 0.12, 2, 4, 8, 24 4 MOD-4023 6# i.v. Male 108 300 43.2/3 0.12,2, 4, 8, 24, 48, 72 Volume of blood sample/time point - 500 μl Terminalblood samples #3 rats per time point.

A commercial sandwich ELISA kit specific for detection of human growthhormone (Roche Diagnostics) was used for evaluation of the rat plasmasamples. This kit detects human growth hormone in plasma by means of anantibody sandwich ELISA format. This kit was initially used to measurethe concentration of MOD-4023 in rat plasma. For these plasma samples, aMOD-4023 standard curve (1.2-100 ng/ml) was used, and the concentrationsof MOD-4023 in rat plasma were interpolated from this curve.

Standard pharmacokinetic parameters, including clearance (CL or CL/F),volume of distribution (Vd or Vd/F), half-life (t_(1/2)), area under theplasma concentration versus time curve (AUC), maximal observed plasmaconcentration (C_(max)) and time to maximal observed plasmaconcentration (T_(max)), were obtained from plasma albutropin or GHconcentration/time curves by noncompartmental analysis using themodeling program WinNonlin (Pharsight, version 3.1). Plasma MOD-4023 orGH concentration data were uniformly weighted for this analysis. The AUCwas calculated using the log-linear trapezoidal analysis for the i.v.data and the linear-up/log-down trapezoidal method for the s.c. data.Plasma concentration profiles for each rat (with the exception of thes.c. albutropin data) or monkey were analyzed individually, andmean±standard error of the mean (S.E.M.) values for the pharmacokineticparameters are reported in Table 13, and FIG. 17.

Single Dose/Repeated Dose Weight Gain Assay (WGA)

Hypophysectomized (interaural method) male rats, 3-4 weeks of age, wereobtained from CRL Laboratories. During a post-surgical acclimationperiod of 3 weeks, rats were examined and weighed twice weekly toeliminate animals deemed to have incomplete hypophysectomy evidenced byweight gain similar to that of sham-operated rats. Those rats withincomplete hypophysectomized were eliminated from the study. The averagebody weights of the hypophysectomized were 70-90, at the time of theexperiment. This is the standard USP and EP bioassay for hGH.Hypophysectomized rats (rats from which the pituitary gland was removed)lose their ability to gain weight. Injections of hGH (and of MOD-4023)to these rats result in weight gain. Based on the measured weight gainalong a defined period of time and the amount of hGH injected, thespecific activity of hGH (and MOD-4023) is determined. Rats wereadministered either a single s.c. dose of 0.4, 0.8 and 4 mg/Kg orrepeated s.c. doses of 0.6 and 1.8 mg/Kg 4 days apart for 3 weeks.Individual body weights of all animals are determined at randomization,prior to the first dosing, thereafter every two days or in case ofdecedents at the time of death, and prior to sacrifice.

Pharmacodynamics/Pharmacokinetics Studies

Hypophysectomized (interaural method) male rats, 3-4 weeks of age, wereobtained from CRL Laboratories. During a post-surgical acclimationperiod of 3 weeks, rats were examined and weighed twice weekly toeliminate animals deemed to have incomplete hypophysectomy evidenced byweight gain similar to that of sham-operated rats. Those rats withincomplete hypophysectomized were eliminated from the study. The averagebody weights of the hypophysectomized and sham rats were 70 and 150 g,respectively, at the time of the experiment.

Rats were administered a single s.c. with MOD-4023, vehicle, humangrowth hormone MOD-4023 or human growth hormone (20 μg/rat) in aninjection volume of 0.2 ml/rat. The dose of GH was 0.35 and 1.05 μg/Kg,a dose of growth hormone that was equimolar with the amount of GH in acorresponding 0.6 and 1.8 μg/Kg dose of MOD-4023. The treatment groupsare summarized in Table 12. Following injection, plasma samples forIGF-1 analyses were obtained at the times described in Table 12. Sampleswere analyzed for IGF-1 concentration using a commercial ELISA (R&Dsystems).

TABLE 12 Treatment schedule for hypophysectomized rat study No. ofanimals/ Eq. Eq. MOD-4023 Dose Trt. Test group/ Dose Dose Dosage Conc.Vol. Time-Points * Grp. Article timepoint Route (mg/rat) (rag/Kg) mg/ml(ml) (hours post-dose) M7 Biotropin ® 9 s.c. 0.032 0.35 0.16 0.2 0(Pre-dose) 0.5, 2, 4, 8, 24, 48, 72, 96 M8 Biotropin ® 9 s.c. 0.095 1.050.475 0.2 0 (Pre-dose) 0.5, 2, 4, 8, 24, 48, 72, 96 M9 EN648-01- 12 s.c.0.032 0.35 0.275 0.2 1, 2, 4, 8, 24, 48, 72, 96 08-005 (0.055) (0.6) M10EN648-01- 12 s.c. 0.095 1.05 0.825 0.2 1, 2, 4, 8, 24, 48, 72, 96 08-005(0.165) (1.8) Volume of blood sample/time point - 500 μl Terminal bloodsamplesCarbohydrate Content and Sialic Acid Content Analysis of O-glycans isbased on a Prozyme kit. O-glycans are chemically and enzymaticallycleaved from the protein and separated from peptides using paperchromatography. Sequencing of the O-glycan pool is performed bysequential enzymatic digestions (exo-glycosidases) followed by HPLCanalysis compared to standards.

Glycoprofiling with Sequence Analysis

Glycoprofiling was performed by Ludger Ltd. Two samples (EN648 andRS0708) were taken through triplicate releases and each release was alsoanalyzed by HPLC in triplicate. Triplicate 300 μg samples of EN648 andRS0708 and a single 1001 sample of citrate/sodium chloride buffer, plusa positive control fetuin (250 μg) and a 100 μl water negative control,were ultra-filtrated by centrifugation using a molecular weight cut offmembrane of 10,000Da to replace the buffer with water, then takenthrough hydrazinolysis under O-mode conditions (6 h at 60° C.). Releasedglycans were re-N-acetylated and cleaned up by LudgerClean CEXcartridges. An aliquot of the released glycans was then labeled with2-aminobenzamide (2AB), cleaned up with Ludger Clean S cartridges andanalyzed by LudgerSep-N2 HILIC-HPLC.

Monosaccharide Content

Analysis of neutral monosaccharides requires hydrolysis of glycans totheir constituent monosaccharide components. The hydrolysis wasperformed by Ludger Ltd, on intact glycoprotein samples. Specifically,50 μg of intact glycoprotein was acid hydrolyzed, 2-AB(2-aminobenzamide) labeled and run on a reverse phase HPLC column. Thismethod hydrolyzes all glycans present on the glycoprotein inclusive of Nand O linked types.

Sialic Acid Profiling

Two samples (EN648 and RS0708) and a buffer control were analyzed.Sialic acid analysis requires mild acid release of the monosaccharidesfollowed by DMB fluorophore labeling and HPLC analysis on a LudgerSep-R1column. 50 μg of intact glycoprotein was acid hydrolyzed for eachanalysis.

Results and Conclusions

MOD-4023 (CTP-hGH-CTP-CTP) is a single chain protein of 275 amino acidsand up to twelve O-linked carbohydrates. The structure consists ofmodified human Growth Hormone (Somatropin) attached to three copies ofthe C-terminal peptide (CTP) of the beta chain of human ChorionicGonadotropin (hCG); one copy at the N-terminus and two copies (intandem) at the C terminus. Human Growth Hormone is comprised of 191amino acids. CTP is comprised of 28 amino acids and four O-linked sugarchains.

Pharmacokinetics of MOD-4023 in SD Rats

The pharmacokinetics of MOD-4023 was evaluated and compared to that ofcommercial hGH (Biotropin®).

TABLE 13 Mean pharmacokinetic parameters following single- dose i.v. ands.c. administration of MOD-4023 and GH (Biotropin ®) in Sprague-Dawleyrats PK Statistics s.c. i.v. MOD- MOD- Parameters Units Biotropin ® 4023Biotropin ® 4023 Dose mg/Kg 50 50 50 50 AUClast hr*ng/mL 41 680 162.71568.3 Cmax ng/ml 13 36.8 275.8 926 Tmax hr 0.5 8 0 0 MRT hr 2.5 12.90.5 9.9 T_(1/2) alpha hr 1.58 0.74 T_(1/2) beta hr 1.73 9 0.5 6.9

Data Statistical Analysis

Analysis of serum samples was performed in order to determine specificconcentration levels for each sample. Concentration and time-point datawere processed using WinNonLin noncompartmental analysis.

Parameters that were determined included: AUC, MRT, t_(1/2), C_(max),and T_(max). FIG. 17 demonstrates the superior pharmacokinetic profileof MOD-4023 plasma concentration compared to GH concentrations (pg/ml)following a single i.v. or s.c. dose of MOD-4023 or GH in rats (n=3-6per dose/route).

Following a single S.C. injection of 50 μg/kg, clearance of MOD-4023from SD rat's blood was significantly slower than that of MOD-4026 andof Biotropin®. The corresponding calculated half-life times and AUCswere:

Biotropin ® T_(1/2) 1.7 h, AUC 41 hr*ng/mL MOD-4026 T_(1/2) 8.5 h, AUC424 hr*ng/mL MOD-4023 T_(1/2) 9.0 h, AUC 680 hr*ng/mL

Conclusion: MOD-4023 was chosen as the final candidate out of 6 othervariants. MOD-4023 demonstrated superior performance in terms ofbiological activity and pharmacokinetics.

Single Dose and Repeated Dose Weight Gain Assay

The results comparing whole body growth response following differentdosing patterns of MOD-4023 in hypophysectomized rats are demonstratedin FIG. 18. The results demonstrate that a single injection of 0.4 & 0.8mg/Kg/day doses of hGH-CTP were equivalent to 4 daily injections of 0.1mg/Kg/day of Biotropin®. The peak of the hGH-CTP effect was after 2days.

FIG. 19 further demonstrates that the area under the curve following asingle injection of MOD-4023 correlates with body weight gain in rats.Thus, the collective data demonstrates that body weight gain is closelycorrelated with cumulative AUC.

hGH-CTP construct administered 4 days apart promotes the same weightgain as daily injections of Biotropin® as demonstrated in FIG. 20. Thehalf-life of hGH in humans is expected to be 5× better than in rats,indicating a potential peak effect in humans after 10 days for onesingle injection. These results support administration of hGH-CTPconstruct, MOD-4023, once weekly or bi-weekly in humans.

Glyco Analysis of MOD-4023

TABLE 14 Glycan analysis. Structural assignments and percentage areas ofpeaks are based upon HPLC and enzyme array digests. Percent from totalglycans^(e) Peak ABS ID^(a) GU^(b) Structure^(c) name Und^(d) NAN1 ABSBTG 1^(f) 0.92 ♦—2AB +bgd GalNac 0.4 0.4 0.6 53.0 2^(f) 1.02 ⋄—2AB +bgdgalactose 1.9 9.7 23.8 26.5 * 1.72 4.3 4.6 2.3 3 1.79

Galβ1-3GalNAc 2.3 67.7 69.4 17.1^(h) 4^(g) 2.25

NeuNacα2-3Gal 19.8 13.0^(h) * 2.57 1.5 1.9 1.1 1.1 5 2.90

NeuNAcα2-3Galβ1-3 GalNac 70.6 * 3.58 0.6 0.7 0.6 6 3.22

Galβ1-3[NeuNAcα2-6] GalNAc 0.9 2.3 7 4.42

NeuNAcα2-3Galβ1-3 [NeuNAcα2-6]GalNAc 1.8

The monosaccharide profiles indicate that the MOD-4023 glycoproteinsamples contain predominantly O-link type glycans. The major O-glycanpeak is sialylated core 1 (Neu5Acα2-3Galβ1-3GalNAc). The major sialicacid is Neu5Ac and there are some minor peaks suggesting the presence of3-4% of di-acetylated sialic acid N-acetyl-9-O-acetylneuraminic acid(Neu5, 9Ac2) and less than 1% N-glycolylneuraminic acid. There are alsosmall amounts of Neu5Acα2-6(Galβ1-3)GalNAc.

Pharmacokinetics and Pharmacodynamics of MOD-4023 in HypophysectomizedRats

Non-compartmental pharmacokinetic analysis was performed on the meanserum concentration versus time curves for each group. MOD-4023 C_(max)was significantly higher than Biotropin® C_(max). The terminal half-lifeof MOD-4023 was 6 times higher than Biotropin®.

TABLE 15 Pharmacokinetic Parameter Estimates of MOD-4023 and Biotropin ®Following a Single Subcutaneous Injection in Hypophysectomized Rats DoseCmax Tmax AUC_(0-∞) AUC_(0-t) CL/F T_(1/2) Group mg/kg Gender ng/mL hrng-hr/mL ng-hr/mL mL/hr/kg hr MOD- 1.8 M 2,150 8 37,713 37,695 0.9286.86 4023 0.6 M 681 8 11,505 11,489 3.042 6.8 hGH 1.05 M 1,078 0.5 3,5413,540 9.884 1 0.35 M 439 0.5 1,279 1,279 27.36 1

The AUC_(0-t) and the AUC_(0-∞) were very similar suggesting theduration of sampling was adequate to characterize the pharmacokineticprofiles. AUC of MOD-4023 was 10 times higher than of Biotropin®.Moreover, C_(max) was generally proportional to dose and for MOD-4023,it was twice as high as the C_(max) of Biotropin®. However, as shown inFIG. 21, T_(max) of MOD-4023 was 8 hr as compare to 0.5 hr of Biotropin®and the terminal half-lives did not appear to vary with dose level. TheT_(1/2) of MOD-4023 was 6.8 times longer than of Biotropin®.

Indirect effects of GH are mediated primarily by an insulin-like growthfactor-I (IGF-I), a hormone that is secreted from the liver and othertissues in response to growth hormone. A majority of thegrowth-promoting effects of growth hormone is actually due to IGF-Iacting on its target cells. Accordingly, the effect of the CTP-hGHconstruct, MOD-4023, on IGF-1 serum levels in hypophysectomized rats wasmeasured. FIG. 22 presents results of IGF-1 serum levels inhypophysectomized rats following s.c. injection of MOD-4023 andcommercial hGH.

A single dose of MOD-4023 0.6 or 1.8 mg/Kg, or Biotropin® 0.35 or 1.05mg/Kg was injected subcutaneously to hypophysectomised rats for thedetermination of the PK/PD profile. Serum IGF-I post injection wasmeasured using specific ELISA kits (Roche Diagnostics).

The cumulative serum levels of IGF-I following injection of MOD-4023 wassignificantly higher than following injection of Biotropin®. The C_(max)was generally proportional to the dose, and for MOD-4023, it was 3-4times higher than C_(max) of Biotropin®. The T_(max) of MOD-4023 was36-48 hr as compared to 20-24 hr of Biotropin®. In conclusion, higherhGH levels and longer presence in serum result in a significant increasein IGF-1 levels.

Pharmacokinetic/Toxicokinetic Analysis in Rhesus Monkeys

Serum concentrations versus time curves were generated for each animal.Non-compartmental analysis was performed with WinNonlin professionalversion 5.2.1 (Pharsight Corporation, Mt View Calif.). The estimatedpharmacokinetic parameters are shown in the table 16 below:

TABLE 16 Estimates of MOD-4023 Pharmacokinetic Parameters (Mean ± SD)from Non-compartmental Analysis Following A Single SubcutaneousInjection in Rhesus Monkeys Parameter 1.8 mg/kg 90 mg/kg Cmax (μg/mL) 2.073 ± 0.417 108.7 ± 46.0 Tmax (hr)  4 ± 0 11 ± 7 AUC_(0-t) (μg-hr/mL)38.7 ± 7.4 2,444 ± 394  AUC_(0-∞) (μg-hr/mL) 39.0 ± 7.3 2,472 ± 388 CL/F (mL/hr/kg) 47.5 ± 9.0 37.04 ± 4.78 T_(1/2) (hr) 10.00 ± 1.47  9.85± 1.07 Vz/F (mL/kg)  701 ± 236  529 ± 104

The AUC_(0-t) and the AUC_(0-∞) were very similar suggesting theduration of sampling was adequate to characterize the pharmacokineticprofiles. The C_(max) was proportional to dose. The T_(max) was later atthe higher dose. The T_(max) was at 4 hours for all animals in the lowdose group and was at 8 or 24 hours in the high dose group. The terminalhalf-lives were similar for the two dose groups.

The AUC was approximately proportional to dose with a slightly largerthan proportional AUC at the higher dose producing a slightly lowerestimate for CL/F and Vz/F compared to the lower dose. It is notpossible to say if CL and Vz are lower at the higher dose or if F islower at the lower dose. There was overlap between the groups so it isquestionable that this represents a meaningful difference in CL/F andVz/F.

Pharmacokinetic parameters estimated by the model were very similar tothose from non-compartment analysis. Absorption and eliminationhalf-lives are shown in Table 17 below:

Table 17: Estimates of MOD-4023 Absorption and Elimination Half-lives(Mean±S. Dak.) Following Subcutaneous Injection Derived FromPharmacokinetic Modeling in Rhesus Monkeys

Dose T_(1/2 abs) (hr) T_(1/2 el) (hr) 1.8 mg/kg  1.17 ± 0.40 10.41 ±2.36 90 mg/kg 6.49 ± 1.87  7.26 ± 1.85

The data indicate that the elimination rates are fairly similar betweenthe groups with a slightly longer T_(1/2) el in the lower dose group.The absorption, however, is more than 5-fold slower followingsubcutaneous administration of 90 mg/kg compared to that following 1.8mg/kg. As in the case of the non-compartmental analysis, modelingindicated a later T_(max) at the high dose.

Although GH supplementation is effective in the treatment of GHdeficiency in children and adults, the disadvantages of daily injectionsover extended periods of time limit its use by physicians in certainpatient populations as well as increase the risk of dosing error, thenumber of care givers, the cost of treatment and noncompliance.Especially important in certain populations, such as children of shortstature who may not understand the implications of not following theprescribed GH dosing regimen, is the necessity of compliance to achievethe optimal benefit from GH therapy. The issue of finding a moresuitable alternative to daily GH injections and subsequent compliancegains further importance as GH-deficient children transition into adultswith a continuing need for GH treatment. The requirement of dailytherapy is largely due to recombinant GH's short plasma half-life andhas led to the development of a sustained release form of GH (Reiter EO. Attire K M., Mashing T J. Silverman B L. Kemp S F. Neolith R B. FordK M. and Sanger P. A multimember study of the efficacy and safety ofsustained release GH in the treatment of naive pediatric patients withGH deficiency. J. Clin. Endocrinol. Metab. 86 (2001), pp. 4700-4706.).

MOD-4023, a recombinant human growth hormone-CTP fusion protein, asdescribed herein, has a pharmacokinetic profile in the rat that islonger in duration than that of GH. This unique pharmacokinetic profileallows for intermittent administration of MOD-4023 to achievepharmacodynamic effects in growth hormone-deficient rat as evidenced bygrowth and elevations in plasma IGF-1 levels, respectively.

MOD-4023 offers a superior pharmacokinetic profile compared with that ofGH when administered s.c. in the rat. There are substantial differencesin plasma clearance of MOD-4023 compared to GH. Specifically, plasma iscleared of MOD-4023 at more than 6 times slower rate than of GHfollowing s.c. dosing. The terminal half-life and mean residence time ofMOD-4023 were approximately six times longer than that of GH in ratsfollowing s.c. administration. In addition, the Cl/F following s.c.dosing is 10 times lower for MOD-4023 than for GH.

In an effort to examine whether the pharmacokinetic advantages in therat translated to a pharmacodynamic benefit, the possibility thatMOD-4023 might stimulate growth in GH-deficient hypophysectomized ratswith dosing regimens less frequent than daily was tested at equimolarMOD-4023 and GH dose levels. Single s.c. injection of MOD-4023 promotedto incremental weight gain which was equal to 4 daily consecutiveinjections of GH. In addition, the every fourth day administrationschedule for MOD-4023 shows enhanced body weight gain over GH.

Pharmacodynamically, the long circulation time of MOD-4023 relative toGH in the hypophysectomized rats resulted in a prolonged IGF-1 responsemeasured in blood plasma following a single s.c. injection. Subcutaneousadministration of a single dose of MOD-4023 increased circulating IGF-1concentrations in a dose-dependent manner in the hypophysectomized rats.At the highest albutropin dose, IGF-1 concentrations were elevated abovebaseline for as long as 75 hours after a single administration. Thus,the enhanced circulation time of a single dose of MOD-4023 resulted insubstantial pharmacodynamic improvement over a single dose of GH,raising the possibility that MOD-4023 could offer similar growthenhancement with reduced dosing frequency compared with standard GHtreatment regimens.

Single CTP-modified hGH doses of 90 mg/kg in rhesus and 180 mg/kg inrats were well tolerated in both species. The allometric factor betweenrats and primates is approximately X2 which is based on the anticipatedclearance of proteins in these animals. In line with industry-acceptedextrapolation models for therapeutic proteins' half-life increasebetween species (FDA Guidance), 90 mg/kg in primates has a PK profileslightly better than 180 mg/kg of CTPs modified hGH in rat. Thus,allometric extrapolation to humans supports weekly or once/2 weekinjections.

The present concept utilizing a CTP-GH construct, reduced the dosingfrequency compared to the commercial GH recombinant product. NutropinDepot® is a sustained release formulation of GH approved for use inpediatric populations; however, comparisons to historical controls haverevealed that 1- and 2-year growth rates are significantly (p<0.001)lower in children given Nutropin Depot® (1-year growth rate 8.2±1.8cm/year) than in children treated with GH (one-year growth rate 10.1±2.8cm/year) (Silverman B L. Blethen S L. Reiter E O. Attie K M. Neuwirth RB. and Ford K M. A long-acting human growth hormone (Nutropin Depot®):efficacy and safety following two years of treatment in children withgrowth hormone deficiency. J. Pediatr. Endocrinol. Metab. 15 (2002), pp.715-722.). The local effects of subcutaneously administered NutropinDepot® include nodules, erythema, pain at the injection site, headacheand vomiting. Preclinical toxicology studies in both rat and monkey haveshown that s.c. administration of CTP-hGH-CTP-CTP produces no localreactions compared to vehicle. Given the medical need for a lessfrequently administered form of GH, the pharmacologic properties ofMOD-4023 in this study in rats suggest that this product is favorablealso in terms of toxicology and patient compliance. The sustainedactivity of MOD-4023 in the rat support its potential utility as anagent that requires only intermittent administration to attain atherapeutic benefit that is currently achieved with daily dosing.

Example 10 Construction of hIFN Beta-CTP Variants

Construction of hIFINβ-CTP variants: A cassette gene containing theC-Terminal peptide (CTP) of the beta subunit of hCG was fused to thecoding sequence of human IFN beta 1a (SEQ ID NO: 49) at differentlocations. Seven IFNβ-CTP variants were constructed as illustrated inFIGS. 1A-G. The proIFNβ signal peptide was used for the construction ofthe secreted IFNβ-CTP variants. XbaI-NotI fragments containing IFNβsequences were ligated into the pCI-dhfr expression vector of thepresent invention.

Table 18 hereinbelow summarizes the primer sequences used forconstructing the CTP-containing polypeptides of the present invention.

TABLE 18 Restriction SEQ site Primer ID (underlined number NO Sequencein sequence) 40 20 5′ GAATTCTAGAGGACATGACCAAC 3′ XbaI 41R 21 5′GCGGCCGCGGACTCATCAGTTCCTCAGGTAGCCG NotI 3′

IFNβ-1 901-1-p107-2 (IFNβ-1—SEQ ID NO: 53): The IFNβ-ctp clone wassynthesized by GeneArt (Geneart No. 0609229).

Then the XbaI-NotI fragment containing IFNβ-ctp sequence was ligatedinto pCI-dhfr expression vector. The amino acid sequence of this cloneis presented in SEQ ID NO: 52.

IFNβ-2 901-2-p113-3 (IFNβ-2—SEQ ID NO: 55): The XbaI/ApaI fragment(IFN-ctp) of pCI-dhfr-701-2-p24-2 (IFN-ctpx2) was replaced by theXbaI/Apa1 fragment (IFNβctp) of 901-1-p107-2 to create a IFN{tilde over(β)}ctpx2 clone. The amino acid sequence of this clone is presented inSEQ ID NO: 54.

IFNβ-4 901-4-p108-16 (IFNβ-4—SEQ ID NO: 59): The ctp-IFNβ-ctp-IFN

clone was synthesized by GeneArt (Geneart No.0609227).

Then the XbaI-NotI fragment containing sequence ctp-IFNβ-ctp-IFNβ wasligated into pCI-dhfr expression vector. The amino acid sequence of thisclone is presented in SEQ ID NO: 11.

IFNβ-6 901-6-p109-3 (IFNβ-6 SEQ ID NO:16): The ctp-IFNβ-ctp clone wassynthesized by GeneArt (Geneart No. 0609228).

Then the XbaI-NotI fragment containing sequence ctp-IFNβ-ctp was ligatedinto pCI-dhfr expression vector. The amino acid sequence of this cloneis presented in SEQ ID NO: 62.

IFNβ-5-p103-10 (IFNβ-5 SEQ ID NO: 14—(ctp-IFNβ): Primers were orderedfrom Sigma-Genosys. A PCR reaction was performed using primer 40 (SEQ IDNO: 65) and primer 41R (SEQ ID NO:21) and plasmid DNA of the synthesizedctp-IFNβ-ctp (Geneart No. 0609228) as a template; as a result of the PCRamplification, a 677 bp product was formed. The PCR fragment wasdigested with XbaI-Nod, and the fragment containing ctp-IFNβ sequencewas ligated into our eukaryotic expression vector pCI-dhfr to yield the901-5-p103-10 clone. The amino acid sequence of this clone is presentedin SEQ ID NO: 60.

IFNβ-3 90′-3-p114-5 (IFNβ-3 SEQ ID NO: 57—(ctp-IFN-CTP(×2)): TheXbaI/ApaI fragment (IFN-ctp) of pCI-dhfr-701-2-p24-2 (IFN-ctpx2) wasreplaced by the XbaI/ApaI fragment (ctp-IFNβ-ctp) of 901-6-p109-3 tocreate a ctp-IFNβctpx2 clone. The amino acid sequence of this clone ispresented in SEQ ID NO: 56.

IFNβ-901-O-p102-1 (IFNβ-0 SEQ ID NO:2-(IFNβ): Primers were ordered fromSigma-Genosys. A PCR reaction was performed using primer 40 (SEQ IDNO:20) and primer 41R (SEQ ID NO:21) and plasmid DNA of the synthesizedIFNβ-ctp (Geneart No. 0609229) as a template; as a result of the PCRamplification, a 599 bp product was formed. The PCR fragment wasdigested with XbaI-NotI, and the fragment containing IFNβ sequence wasligated into our eukaryotic expression vector pCI-dhfr to yield the901-O-p102-1 clone. The amino acid sequence of this clone is presentedin SEQ ID NO: 1.

Example 11 Expression and Isolation of IFN-CTP Polypeptides Materialsand Methods

DNA transfection and clone selection: DG44 cells were transfected withpCI-DHFR expression vectors containing IFNβ-CTP variants using FuGENE6Reagent (FuGENE Transfection Reagent—Roche Cat.11 815 091 001).Forty-eight hr following transfection, cells were diluted and seeded at50-200 cells per well in a selective medium (CD DG44 Medium w/o HT(Gibco: Scotland part: #07990111A) Sku num.:ME060027 supplemented with 8mM L-Glutamine Biological Industries: Cat: 03-020-1A) and 18 mL/L of 10%Pluronic F-68 solution (Gibco: Cat: 240040-032). Selected clones werescreened for highest protein production using commercial ELISA. Three tofive producing clones per each variant were frozen for a backup cellbank. A selected clone for each variant was adapted to growth in largerscale cultures up to 1L flasks on an orbital shaker platform.Supernatants were collected and analyzed by ELISA, SDS-PAGE and Westernblot. Following the withdrawal of aliquots, the protein-containingsupernatants were kept frozen until further use.

Cell culture: DG44 cells were maintained in DG44 medium with HT(cat#12610-010, Gibco) supplemented with 8 mM L-Glutamine (BiologicalIndustries: Cat: 03-020-1A) and 18 mLJL of 10% Pluronic F-68 solution(Gibco: Cat: 240040-032), at 37° C. in humidified 8% CO₂ incubator.Transfected clones were maintained in DG44 basal medium without HTsupplement, hypoxanthine and thymidine, with pluronic acid andL-glutamine.

Sample preparation: Supernatants were collected, filtrated and analyzedby ELISA to determine protein concentration. SDS-PAGE and Western blotwere used to determine purity and identity. Following ELISA, sampleconcentrations were defined and the solution was dialyzed against PBS.Following the withdrawal of aliquots, the protein-contained supernatantswere kept frozen at −20° C. until further use.

Western Blotting: Samples were electrophoresed on nondenaturing 15SDS-polyacrylamide gels. Gels were allowed to equilibrate for 10 min in25 mM Tris and 192 mM glycine in 20% (vol/vol) methanol). Proteins weretransferred to a 0.2 μm pore size nitrocellulose membrane (Sigma, SaintLouis, Mo.) at 250 mA for 3 h, using a Mini Trans-Blot electrophoresiscell (Biorad Laboratories, Richmond, Calif.). The nitrocellulosemembrane was incubated in 5% non-fat dry milk for 2 h at roomtemperature. The membrane was incubated with IFN anti-serum (1:1000titer) overnight at 4° C. followed by three consecutive washes in PBScontaining 0.1% Tween (10 min/wash). The membrane was incubated withsecondary antibody conjugated to Horse Radish Peroxidase (HRP) (Zymed,San Francisco, Calif.) for 2 h at room temperature, followed by threewashes. Finally, the nitrocellulose paper was reacted with enhancedchemiluminescent substrate (ECL) (Pierce, Rockford, Ill.) for 5 min,dried with a Whatman sheet, and exposed to X-ray film.

FIG. 12 indicates that MOD-901X-variants are recognized by anti IFN-β1aantibodies. The SDS PAGE gel was stained using coomassie blue (A) or (B)blotted and stained using monoclonal anti-IFNβ1a antibodies.

Example 12

The IFN-CTP Polypeptides are Bioactive

To determine the bioactivity of MOD-901X variants through itsrecognition and binding to the IFN receptor, the Daudi cell line (humanBurkitt lymphoma, ATCC catalog No, CCL-213 ™), one of the most sensitivecell lines to the anti-proliferative effect of IFN-β1a were used. Daudicells, grown in suspension were treated with different concentrations ofIFN-β1a (50-1000 μg/ml final concentration) and incubated for 72 hours.The number of viable cells was measured using CellTiter 96® AQueous OneSolution Cell Proliferation Assay kit (Promega G3580) according tomanufacturer procedures. The assay's standard curve was prepared usingrecombinant human IFN-β1a (PtoSpec Tany TechnoGene).

IFN-β1a is a cytokine that exhibit antiviral activity against a varietyof viruses. The potency of IFN-β1a as an antiviral agent can bedetermined by a viral cytopathic effect (CPE) bioassay that measures theability of the protein to protect human lung carcinoma A549 cells (grownat 37° C., 5% CO₂) challenged with encephalomyocarditis (ECM) virus.A549 cells were plated into 96 well microtiter plate. Serial dilutionsof IFN-β1a standards and test samples were added, and 24 h later, thecells were challenged with ECP virus. Viable cells were quantified twodays later.

The potency (titer) of an IFN-β1a test sample is determined as thereciprocal of the dilution represented in the well in which 50% of thecell monolayer is protected from the CPE virus. The actual potency iscalculated by comparing the sample's protective effect with the sameeffect of a reference standard calibrated in International Units,provided by the National Institute of Allergy and Infectious Diseases(NIH). The results are shown in Table 19.

TABLE 19 Specific Activity IU/mg × 10{circumflex over ( )}8 Anti-viralAnti-proliferation IC50 pg/ml Intl. Standard 2.00 318 IFNb-0 3.90 2.53251 IFNb-1 4.00 2.41 264 IFNb-2 4.00 1.90 334 IFNb-3 4.00 2.77 230IFNb-5 4.00 6.24 102 IFNb-6 3.70 1.97 323 *concentration was determinedby Elisa assay

Conclusion: The activity of MOD-901X variants as measured by itsantiviral effects were at normal range of the international standard andsimilar to rhIFN. The same effect was observed in an anti-proliferationassay except for MOD-9015 which was 3 times more potent than the othervariants. IFNb-0 is SEQ ID NO: 1. IFNb-1 is SEQ ID NO: 52 (MOD-9011).IFNb-2 is SEQ ID NO: 54 (MOD-9012). IFNb-3 is SEQ ID NO: 56 (MOD-9013).IFNb-4 is SEQ ID NO: 58 (MOD-9014). IFNb-5 is SEQ ID NO: 60 (MOD-9015).IFNb-6 is SEQ ID NO: 62 (MOD-9016).

Example 13

Comparative Pharmacokinetics (MOD-901X Variants, Avonex® and Rebif®)

In order to determine the pharmacokinetics of MOD-901X and compare it tothat of commercial IFN-β1a (Rebif®, Avonex®) data statistical analysiswas performed. The analysis included analysis of serum samples that wasperformed in order to determine specific concentration levels for eachsample. Concentration and time-point data were processed using WinNonLinnocomparmental analysis. The following parameters were determined: AUC,CL, Ke, t_(1/2), C_(max), T_(max), and Vdz.

The experimental design is provided in Table 20.

TABLE 20 Equimolar Dose Species/ Dose Vol. Time-Points * No. Drug NRoute Gender (μg/Kg) (ml) (hours post-dose) 1 Avonex 3 IV/SC SD rat/Male38/66 0.3/0.5 0 (Pre-dose) 0.5, 4, 8, 24, 48, 96. 2 Rebif 3 IV/SC SDrat/Male 38/66 0.3/0.5 0.5, 4, 8, 24, 48, 96. 3 MOD-9011 3 IV/SC SDrat/Male 38/66 0.3/0.5 0.5, 4, 8, 24, 48, 96. 4 MOD-9012 3 IV/SC SDrat/Male 38/66 0.3/0.5 0.5, 4, 8, 24, 48, 96. 5 MOD-9013 3 IV/SC SDrat/Male 38/66 0.3/0.5 0.5, 4, 8, 24, 48, 96. 6 MOD-9015 3 IV/SC SDrat/Male 38/66 0.3/0.5 0.5, 4, 8, 24, 48, 96. 7 MOD-9016 3 IV/SC SDrat/Male 38/66 0.3/0.5 0.5, 4, 8, 24, 48, 96.

#3 rats per time point.

FIG. 13 shows the change in serum concentration of IFN-β1a or MOD-901Xconcentrations (ng/ml) following single-dose i.v. administration ofIFN-β1a or MOD-901X in SD rats.

Table 21 shows the mean pharmacokinetic parameters following single-dosei.v. or Sub-Cutaneous (s.c.) administration of IFN-β1a and MOD-901X inSprague-Dawley rats.

TABLE 21 PK Statistics IV Parameters Units Avonex Rebif MOD-9011MOD-9012 MOD-9015 MOD-9013 MOD-9016 Dose μg 5 5 5 5 5 5 5 AUClasthr*ng/mL 83.9 106.4 185.3 417 369.4 2562.9 879.6 Cmax ng/ml Tmax hr MRThr 1.5 1.3 2.1 11.3 2 12.1 9.6 T½ α hr 1.02 0.9 1.43 2.17 1.4 2.53 2.22T½ β hr 7.82 8.36 6.66

# Parameters was generated for individual rats and the mean data arepresented here.

In conclusion: IFN-β1a with 3 CTP units has 8 times longer half-lifethan that of Rebif® or Avonex® when injected i.v.

FIG. 13 shows the mean plasma of Rebif®, MOD-9012, and MOD-9013concentrations (ng/ml) following single-dose i.v. or s.c. administrationof IFN-[3]a, MOD-9012 or MOD-9013 in SD rats (n=3 perdose/route/timepoint). IFN-[3]a serum concentrations were determinedusing commercial ELISA kit.

Table 22 displays the mean pharmacokinetic parameters followingsingle-dose i.v. or s.c. administration of Rebif®, MOD-9012, andMOD-9013 in Spargue-Dawley rats.

TABLE 22 PK Statistics SC IV Parameters Units Rebif MOD-9012 MOD-9013Parameters Rebif MOD-9012 MOD-9013 Dose μg 10 10 10 Dose 5 5 5 AUClasthr*ng/mL 34.8 498.5 2299.5 AUClast 106.4 417 2562.9 Cmax ng/ml 6.6 19.761.1 Cmax Tmax hr 2 8 8 Tmax MRT hr 4.1 15.9 24.1 MRT 1.3 11.3 12.1 T½ab hr 0.6 2.75 3.1 T½ α 0.9 2.17 2.53 T½ el hr 2.1 9.5 14.2 T½ β 7.828.36

# Parameters were generated for individual rats and the mean data arepresented.

In conclusion, IFN-β1a with 3 CTP units (MOD-9013) has 9.2 times longerhalf-life than that of Rebif® when injected i.v. and 6.7 times longerhalf-life when injected s.c. AUClast of MOD-9013 is 66 times better thenRebif® when injected s.c. and 24 times better when injected i.v. MRT ofMOD-9013 is 5.8 times better when injected s.c. and 9.3 times betterwhen injected i.v.

The MOD-9013 molecule, which comprises one CTP attached to theN-terminus of IFN-β1a and two CTP attached to its C-terminus, was testedin vitro for its ability to bind to the human IFN receptor and in vivofor its pharmacokinetic performance. The conclusions of these studiescan be summarized as follows: (1) The in vitro anti-proliferationactivity of MOD-9013 as demonstrated in the Daudi cell assay was similarto the international standard and to that of MOD-9010 (rIFN-β1aexpressed by Modigene). (2) The anti-viral protective activity ofMOD-9013 shown in Daudi cells was the same as the international standardand as of that of MOD-9010 (rIFN-β1a expressed by Modigene). (3) Interms of its pharmacokinetic features, MOD-9013 was compared in SD ratsto Rebif® and Avonex®. Following a single i.v./s.c. injection of 38/66μg/kg, clearance of MOD-9013 from SD rats blood was significantly slowerthan that for Rebif® and Avonex®. The corresponding calculated half lifetimes and AUCs for i.v. administration were:

Rebif ® T_(1/2), 1 h, AUC106 hr*ng/mL MOD-9013 T_(1/2) 8.4 h, AUC2563hr*ng/mL

For s.c. administration, the corresponding calculated half life timesand AUCs were:

Rebif ® T_(1/2) 2.1 h, AUC34.8 hr*ng/mL MOD-9013 T_(1/2) 14.2 h,AUC2299.5 hr*ng/mL

The superior performance of MOD-9013 to stimulate anti-viral andanti-proliferation activity and to retain long lasting stimulationresults from three main reasons: i) Addition of up to 24 sialic acidresidues; ii) Stabilizing effect on the IFN-β1a molecule by fusing theCTP cassettes to both N and C termini; and iii) Increase in molecularweight of the whole molecule to from ˜31,242-48,000 Daltons.

As shown hereinabove, different levels of potency were exerted byIFN-CTP polypeptides, indicating differences in receptor binding.IFN-CTP polypeptides differ by the number of CTP cassettes and thelocation to which they are fused. MOD-9011 and MOD-9012 contain 1 CTPsequence or 2 CTP sequences at the C-terminal of IFN protein, whileMOD-9013 contains 1 CTP at N-terminal and 2 CTP sequences at C-terminal.MOD-9014 is a dimer of two IFN molecules linked by CTP sequence.MOD-9013 demonstrated unexpected potency level.

1.-24. (canceled)
 25. A polynucleotide comprising a nucleic acidsequence encoding a polypeptide, said polypeptide consisting of acytokine, one chorionic gonadotrophin carboxy terminal peptide (CTP)attached to the amino terminus of said cytokine, and two chorionicgonadotrophin CTPs attached to the carboxy terminus of said cytokine.26. The polynucleotide of claim 25, wherein at least one CTP isglycosylated.
 27. The polynucleotide of claim 25, wherein said at leastone CTP is truncated.
 28. The polynucleotide of claim 25, wherein atleast one CTP is attached to said cytokine via a linker.
 29. Thepolynucleotide of claim 28, wherein said linker is a peptide bond. 30.The polynucleotide of claim 25, wherein the sequence of at least one CTPconsists of an amino acid sequence selected from the group consistingof: SEQ ID NO: 17 and SEQ ID NO:
 18. 31. The polynucleotide of claim 25,wherein said polypeptide further comprises a signal peptide attached tothe amino terminus of said one CTP.
 32. The polynucleotide of claim 31,wherein the sequence of said signal peptide consists of an amino acidsequence selected from the group consisting of: SEQ ID NO: 19 and SEQ IDNO:
 64. 33. The polynucleotide of claim 25, wherein said cytokine is aninterferon.
 34. The polynucleotide of claim 25, wherein said interferonis a type I interferon.
 35. The polynucleotide of claim 34, wherein saidtype I interferon is IFN-α or IFN-β.
 36. The polynucleotide of claim 25,wherein said interferon is IFN-γ.
 37. An expression vector comprisingthe polynucleotide of claim
 25. 38. An isolated cell comprising theexpression vector of claim
 37. 39. A composition comprising theexpression vector of claim
 37. 40. A method of producing a cytokine withan improved biological half life in an isolated cell, comprising thestep of transfecting said cell with an expression vector of claim 37,thereby producing a cytokine with an improved biological half life in anisolated cell.
 41. The method of claim 40, wherein said cytokine is aninterferon (IFN).
 42. The method of claim 41, wherein said IFN is IFN-β.43. A method of producing a cytokine having biological activity in anisolated cell, comprising the step of transfecting said cell with anexpression vector of claim 37, thereby producing a cytokine havingbiological activity in an isolated cell.
 44. The method of claim 43,wherein said cytokine is an interferon.
 45. The method of claim 44,wherein said interferon is IFN-β.